Hairpin loop method for double strand polynucleotide sequencing using transmembrane pores

ABSTRACT

The invention relates to a new method of sequencing a double stranded target polynucleotide. The two strands of the double stranded target polynucleotide are linked by a bridging moiety. The two strands of the target polynucleotide are separated using a polynucleotide binding protein and the target polynucleotide is sequenced using a transmembrane pore.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.14/234,698 filed on Apr. 25, 2014, which is a national stage filingunder U.S.C. § 371 of PCT International Application No.PCT/GB2012/051786, with an international filing date of Jul. 25, 2012,which claims the benefit under 35 U.S.C. § 119(e) to U.S. ProvisionalApplication Ser. No. 61/511,436 filed on Jul. 25, 2011, the contents ofeach of which are incorporated herein by reference in their entireties.

FIELD OF THE INVENTION

The invention relates to a new method of sequencing a double strandedtarget polynucleotide. The two strands of the target polynucleotide arelinked by a bridging moiety. The two strands of the targetpolynucleotide are separated by a polynucleotide binding protein.Sequencing of the target polynucleotide is carried out using atransmembrane pore.

BACKGROUND OF THE INVENTION

There is currently a need for rapid and cheap nucleic acid (e.g. DNA orRNA) sequencing technologies across a wide range of applications.Existing technologies are slow and expensive mainly because they rely onamplification techniques to produce large volumes of nucleic acid andrequire a high quantity of specialist fluorescent chemicals for signaldetection.

Transmembrane pores (nanopores) have great potential as direct,electrical biosensors for polymers and a variety of small molecules. Inparticular, recent focus has been given to nanopores as a potential DNAsequencing technology.

When a potential is applied across a nanopore, there is a drop in thecurrent flow when an analyte, such as a nucleotide, resides transientlyin the barrel for a certain period of time. Nanopore detection of thenucleotide gives a current blockade of known signature and duration. Theconcentration of a nucleotide can then be determined by the number ofblockade events (where an event is the translocation of an analytethrough the nanopore) per unit time to a single pore.

In the “Strand Sequencing” method, a single polynucleotide strand ispassed through the pore and the nucleotides are directly identified.Strand Sequencing can involve the use of a nucleotide handling enzyme,such as Phi29 DNA polymerase, to control the movement of thepolynucleotide through the pore. Nanopore sequencing, using enzymes tocontrol the translocation of dsDNA through the nanopore, has in the pastfocused on only reading one strand of a dsDNA construct. When the enzymeis used as polymerase, the portion to be sequenced is single stranded.This is fed through the nanopore and the addition of dNTPs at aprimer/template junction on top of the strand pulls the single strandedportion through the nanopore in a controlled fashion. The majority ofthe published literature uses this approach to control strand movement(Lieberman et al. (2010) “Processive Replication of Single DNA Moleculesin a Nanopore Catalyzed by phi29 DNA Polymerase” J. Am. Chem. Soc.132(50): 17961-17972). In the polymerase mode, the complementary strandcannot be sequenced. When the enzyme is used as a double strandedexonuclease as published (Lieberman et al. (2010) supra), the unzippingof the complementary strand is accompanied by the digestion of thisstrand. It is therefore not possible to sequence the complementarystrand with this approach. The complementary strand cannot therefore becaptured and sequenced by the nanopore. Hence, only half of the DNAinformation in dsDNA is sequenced.

In more detail, when both polymerase and exonuclease activity areinhibited (by running without tri-phosphates bases and with excess ofEDTA), enzymes such as Phi29 DNA polymerase have been shown to unzipdsDNA when pulled through a nanopore by a strong applied field (FIG. 1)(Lieberman et al. (2010) supra). This has been termed unzipping mode.Unzipping mode implies that it is the unzipping of dsDNA above orthrough the enzyme, and importantly, it is the requisite force requiredto disrupt the interactions of both strands with the enzyme and toovercome the hydrogen bonds between the hybridised strands. In the pastthe second complementary strand was considered to be essential forefficient enzyme binding. In addition, it was thought that the requisiteforce required to unzip the strand above or in the enzyme was a dominantbraking effect slowing DNA through the pore. Herein we describe howenzymes such as Phi29 DNA polymerase can act as a molecular brake forssDNA, enabling sufficient controlled movement through a nanopore forsequencing around the hairpin turns of specially designed dsDNAconstructs to sequence both the sense and anti-sense strands of dsDNA(FIG. 2). Unzipping mode has in the past predominantly been performedusing templates where the distal part of the analyte is blunt ended(FIG. 1). Small hairpins have occasionally been used, but were onlyincluded to simplify DNA design. Previous work has not considered theuse of hairpins on long dsDNA to provide the ability to read bothstrands. This is because the unzipping movement model has not consideredPhi29 DNA polymerase or related enzymes capable of controlling themovement of the DNA when entering ssDNA regions (i.e. when moving aroundthe hairpin and along the anti-sense strand—FIG. 2).

SUMMARY OF THE INVENTION

The inventors have surprisingly demonstrated that both strands of adouble stranded target polynucleotide can be sequenced by a nanoporewhen the two strands are linked by a bridging moiety and then separated.Furthermore, the inventors have also surprisingly shown that an enzyme,such as Phi29 DNA polymerase, is capable of separating the two strandsof a double stranded polynucleotide, such as DNA, linked by a bridgingmoiety and controlling the movement of the resulting single strandedpolynucleotide through the transmembrane pore.

The ability to sequence both strands of a double stranded polynucleotideby linking the two strands with a bridging moiety has a number ofadvantages, not least that both the sense and anti-sense strands of thepolynucleotide can be sequenced. These advantages are discussed in moredetail below.

Accordingly, the invention provides a method of sequencing a doublestranded target polynucleotide, comprising:

-   -   (a) providing a construct comprising the target polynucleotide,        wherein the two strands of the target polynucleotide are linked        at or near one end of the target polynucleotide by a bridging        moiety;    -   (b) separating the two strands of the target polynucleotide to        provide a single stranded polynucleotide comprising one strand        of the target polynucleotide linked to the other strand of the        target polynucleotide by the bridging moiety;    -   (c) moving the single stranded polynucleotide through a        transmembrane pore such that a proportion of the nucleotides in        the single stranded polynucleotide interact with the pore; and    -   (d) measuring the current passing through the pore during each        interaction and thereby determining or estimating the sequence        of the target polynucleotide,    -   wherein the separating in step (b) comprises contacting the        construct with a polynucleotide binding protein which separates        the two strands of the target polynucleotide.

The invention also provides:

-   -   a kit for preparing a double stranded target polynucleotide for        sequencing comprising (a) a bridging moiety capable of linking        the two strands of the target polynucleotide at or near one end        and (b) at least one polymer;    -   a method of preparing a double stranded target polynucleotide        for sequencing, comprising:        -   (a) linking the two strands of the target polynucleotide at            or near one end with a bridging moiety; and        -   (b) attaching one polymer to one strand at the other end of            the target polynucleotide and thereby forming a construct            that allows the target polynucleotide to be sequenced using            a transmembrane pore;    -   a method of sequencing a double stranded target polynucleotide,        comprising:        -   (a) providing a construct comprising the target            polynucleotide, wherein the two strands of the target            polynucleotide are linked at or near one end of the target            polynucleotide by a bridging moiety;        -   (b) separating the two strands of the target polynucleotide            to provide a single stranded polynucleotide comprising one            strand of the target polynucleotide linked to the other            strand of the target polynucleotide by the bridging moiety;        -   (c) synthesising a complement of the single stranded            polynucleotide, such that the single stranded polynucleotide            and complement form a double stranded polynucleotide;        -   (d) linking the two strands of the double stranded            polynucleotide at or near one end of the double stranded            polynucleotide using a bridging moiety;        -   (e) separating the two strands of the double stranded            polynucleotide to provide a further single stranded            polynucleotide comprising the original single stranded            polynucleotide linked to the complement by the bridging            moiety;        -   (f) moving the complement through a transmembrane pore such            that a proportion of the nucleotides in the complement            interact with the pore; and        -   (g) measuring the current passing through the pore during            each interaction and thereby determining or estimating the            sequence of the target polynucleotide, wherein the            separating in step (e) comprises contacting the construct            with a polynucleotide binding protein which separates the            two strands of the target polynucleotide;    -   an apparatus for sequencing a double stranded target        polynucleotide, comprising: (a) a membrane; (b) a plurality of        transmembrane pores in the membrane; (c) a plurality of        polynucleotide binding proteins which are capable of separating        the two strands of the target polynucleotide; and (d)        instructions for carrying out the method of the invention; and    -   an apparatus for sequencing a double stranded target        polynucleotide, comprising: (a) a membrane; (b) a plurality of        transmembrane pores in the membrane; and (c) a plurality of        polynucleotide binding proteins which are capable of separating        the two strands of the target polynucleotide, wherein the        apparatus is set up to carry out the method of the invention.

DESCRIPTION OF THE FIGURES

FIG. 1 shows a schematic of enzyme controlled dsDNA and ssDNAtranslocation through a nanopore. An enzyme (e.g. Phi29 DNA polymerase)that is incubated with dsDNA having an ssDNA leader binds at thessDNA-dsDNA interface. DNA-enzyme complexes are captured by a nanoporeunder an applied field. Under the field, the template strand of the DNAis slowly stripped through the enzyme in a controlled base-by-basemanner, in the process unzipping the complementary primer strand of thedsDNA in or above the enzyme. Once the enzyme reaches the end of thedsDNA it falls off the DNA, releasing it through the nanopore.

FIG. 2 shows another schematic of enzyme controlled dsDNA and ssDNAtranslocation through a nanopore. The dsDNA has a hairpin turn linkingthe sense and anti-sense strands of the dsDNA. Once the enzyme reachesthe hairpin it remains bound to the DNA, proceeds around the hairpinturn, and along the anti-sense strand. In the hairpin and antisenseregions the enzyme functions as an ssDNA molecular brake, continuing tosufficiently control translocation of the DNA through the nanopore tosequence the DNA.

FIG. 3 shows a schematic overview of reading around a hairpin of dsDNAusing the ability of the enzyme to control movement in ssDNA regions.The dsDNA has a 5′-ssDNA leader to allow capture by the nanopore. Thisis followed by a dsDNA section, where the sense and anti-sense strandsare connected by a hairpin. The hairpin can optionally contain markers(e.g. abasic residues, shown in FIG. 3 as a cross) that are observedduring sequencing, which permit simple identification of the sense andanti-sense strands during sequencing. The 3′-end of the anti-sensestrand can optionally also have a 3′-ssDNA overhang, which if greaterthan ˜20 bases allows full reading of the anti-sense strand (theread-head of the nanopore is ˜20 bases downstrand from the top of theDNA in the enzyme).

FIG. 4 shows a schematic of the DNA-Enzyme-nanopore complex (left)sequenced in unzipping mode through MspA nanopores using Phi29 DNApolymerase, and the consensus sequence obtained from them (right).Section 1 marks the sense section of DNA, and section 2 marks theanti-sense section. This figure shows DNA sequencing of a short dsDNAconstruct. In this construct the dsDNA section is not connected by ahairpin, so the enzyme falls off the end of the DNA, and only thetemplate/sense strand is sequenced (except for the last ˜20 bases).

FIG. 5 shows a schematic of the DNA-Enzyme-nanopore complex (left)sequenced in unzipping mode through MspA nanopores using Phi29 DNApolymerase, and the consensus sequence obtained from them (right).Section 1 marks the sense section of DNA, and section 2 the anti-sensesection. DNA sequencing of a short dsDNA construct with a hairpin. Inthis construct the enzyme moves along the sense strand, around thehairpin loop, and down the anti-sense strand, permitting sequencing ofboth the sense and the first part of the anti-sense strand.

FIG. 6 shows a schematic of the DNA-Enzyme-nanopore complex (left)sequenced in unzipping mode through MspA nanopores using Phi29 DNApolymerase, and the consensus sequence obtained from them (right).Section 1 marks the sense section of DNA, and section 2 the anti-sensesection. Similar to FIG. 5, this construct permits sequencing of boththe sense and anti-sense strands, but the additional 3′-ssDNA overhangpermits reading of the full length of the anti-sense strand before theenzyme falls off the end of the DNA.

FIG. 7 shows a schematic of the DNA-Enzyme-nanopore complex (left)sequenced in unzipping mode through MspA nanopores using Phi29 DNApolymerase, and the consensus sequence obtained from them (right).Section 1 marks the sense section of DNA, and section 2 the anti-sensesection. Similar to FIG. 5, this construct permits sequencing of boththe sense and anti-sense strands, however, this construct has a singleabasic residue (shown as a cross) in the hairpin, which provides a clearmarker in the DNA sequence to identify the sense and anti-sensesections.

FIG. 8 shows the consensus DNA sequence of UA02 through MspA. Section 1marks the homopolymeric′ -overhang initially in the nanopore. Section 2marks the sense section of the DNA strand. Section 3 marks the turn.Section 4 marks the anti-sense region of the DNA strand. Thepolynucleotide sequence that corresponds to each section is shown belowthat section number. The sequence shown in this Figure corresponds toSEQ ID NO: 37.

FIG. 9 shows a schematic of a genomic template for unzipping throughMspA nanopores using Phi29 DNA polymerase. It shows a general designoutline for creating dsDNA suitable for reading around hairpins. Theconstructs have a leader sequence with optional marker (e.g. abasic DNA)for capture in the nanopore, and hairpin with optional marker, and atail for extended reading into anti-sense strand with optional marker.

FIG. 10 shows a schematic of the adapter design for ligating ssDNAoverhangs (left) and hairpin turns (right) onto genomic dsDNA. X=abasicDNA. Chol=cholesterol-TEG DNA modification.

FIG. 11 shows typical polymerase controlled DNA movement of a400mer-hairpin through MspA using Phi29 DNA polymerase. Senseregion=abasic 1 to 2. Anti-sense region=abasic 2 to 3.

FIG. 12 shows a consensus DNA sequence profile from multiple polymerasecontrolled DNA movements of a 400mer-hairpin through MspA. Senseregion=abasic 1 to 2. Anti-sense region=abasic 2 to 3.

FIG. 13 shows a schematic of an alternative sample preparation forsequencing. A construct is illustrated comprising the targetpolynucleotide and a bridging moiety (hairpin) linking the two strandsof the target polynucleotide. The construct also comprises a leaderpolymer (a single stranded sequence), a tail polymer (also a singlestranded sequence) and an abasic marker region within the leader. Themarker may prevent the enzyme from making the template completely bluntended i.e. filling in opposite the required leader ssDNA. A stranddisplacing polymerase (nucleic acid binding protein) separates the twostrands of the construct, initiating either via a complementary primeror by protein primed amplification from the tail polymer. A complementis generated to the resulting single stranded polynucleotide. Thecomplement and the original sense and antisense single strandedpolynucleotide analyte can be further modified by addition of a secondbridging moiety (hairpin).

FIG. 14 shows a specific preparation of the construct comprising thetarget polynucleotide.

FIG. 15 shows where amplification may be added as part of the samplepreparation to aid the detection of epigenetic information. A nucleotidehas been constructed so that the following information is read throughthe pore: sense (original), antisense (original), bridging moiety, sense(replicate), antisense (replicate). Information on the methylated base(mC) is therefore obtained four times.

FIG. 16 shows how RNA can be sequenced. A bridging moiety is attached toa piece of RNA and the DNA reverse complement added to the RNA via areverse transcriptase. The RNA is read, followed by the DNA of thereverse complement.

FIG. 17 shows a schematic of helicase controlled dsDNA and ssDNAtranslocation through a nanopore. The dsDNA has a hairpin turn linkingthe sense and anti-sense strands of the dsDNA. Once the enzyme reachesthe hairpin it remains bound to the DNA, proceeds around the hairpinturn, and along the anti-sense strand. In the hairpin and antisenseregions the enzyme functions as an ssDNA molecular brake, continuing tosufficiently control translocation of the DNA through the nanopore tosequence the DNA.

FIG. 18 shows the polynucleotide MONO hairpin construct (SEQ ID NOs: 29to 35) used in Example 4.

FIG. 19 shows a typical helicase controlled DNA movement of a 400 bphairpin (SEQ ID NOs: 29 to 35 connected as shown in FIG. 18) through anMspA nanopore (MS(B1-G75S-G77S-L88N-Q126R)8 MspA (SEQ ID NO: 2 with themutations G75S/G77S/L88NiQ126R)). Sense=region 1. Anti-sense=region 2.

FIG. 20 shows the beginning of a typical helicase controlled DNAmovement of a 400 bp hairpin (SEQ ID NOs: 29 to 35 connected as shown inFIG. 18) through an MspA nanopore (MS(B1-G75S-G77S-L88N-Q126R)8 MspA(SEQ ID NO: 2 with the mutations G75S/G77S/L88N/Q126R)). The polyTregion at the beginning of the sequence is highlighted with a * and theabasic DNA bases as a #.

FIG. 21 shows the transition between the sense and antisense regions ofa typical helicase controlled DNA movement of a 400 bp-hairpin (SEQ IDNOs: 29 to 35 connected as shown in FIG. 18) through an MspA nanopore(MS(B1-G75S-G77S-L88N-Q126R)8 MspA (SEQ ID NO: 2 with the mutationsG75S/G77S/L88N/Q126R)). The transition region between the sense andantisense regions of the sequence is highlighted by a *, the senseregion labeled 1 and the antisense region labeled 2.

FIG. 22 shows an example sample prep method for forming DUO hairpinconstructs. The double stranded DNA analyte is contacted by and modifiedto contain a Y-shaped adapter (the sense strand (SEQ ID NO: 29 attachedto SEQ ID NO: 30 via four abasic DNA bases) of this adaptor contains the5′ leader, a sequence that is complementary to the tether (SEQ ID NO:35, which at the 3′ end of the sequence has six iSp18 spacers attachedto two thymine residues and a 3′ cholesterol TEG) and 4 abasics and theantisense half of the adaptor contains a 3′ hairpin (SEQ ID NO: 31)) atone end of the duplex and a hairpin (SEQ ID NO: 32) at the other. TheY-shaped adapter itself also carries a 3′-hairpin (SEQ ID NO: 31), whichallows extension either by a polymerase or by ligation. This extensionis preferentially carried out by a mesophilic polymerase that has stranddisplacement activity. As the polymerase extends from the 3′ of theY-shaped adapter hairpin (SEQ ID NO: 31) it copies the antisense strand(SEQ ID NO: 34) and so displaces the original sense strand (SEQ ID NO:33). When the polymerase reaches the end of the antisense strand (SEQ IDNO: 34) it fills-in opposite the hairpin (SEQ ID NO: 32) and then beginsto fill-in opposite the now single stranded and original sense strand(SEQ ID NO: 33). Extension is then halted by a section of abasic orspacer modifications (other possible modifications which could haltenzyme extension include RNA, PNA or morpholino bases and iso-dC oriso-dG) to leave the 5′-region of the Y-shaped adapter single stranded(SEQ ID NO: 29).

FIG. 23 shows the specific preparation method used in Example 5 forpreparing a DUO hairpin construct (SEQ ID NOs: 29 to 36 connected asshown in FIG. 25). A ˜400 bp region of PhiX 174 was PCR amplified withprimers containing SacI and KpnI restriction sites (SEQ ID Nos: 27 and28 respectively). Purified PCR product was then SacI and KpnI digestedbefore aY-shaped adapter (sense strand sequence (SEQ ID NO: 29 attachedto SEQ ID NO: 30 via four abasic DNA bases) is ligated onto the 5′ endof SEQ ID NO: 33 and the anti-sense strand (SEQ ID NO: 31) is ligatedonto the 3′ end of the SEQ ID NO: 34) and a hairpin (SEQ ID NO: 32, usedto join SEQ ID NO's: 33 and 34) were ligated to either end, using T4 DNAligase (See FIG. 18 for final DNA construct). The doubly ligated productwas PAGE purified before addition of Klenow DNA polymerase, SSB andnucleotides to allow extension from the Y-shaped adapter hairpin (SEQ IDNO: 31). To screen for successful DUO product a series of mismatchrestriction sites were incorporated into the adapter sequences, wherebythe enzyme will cut the analyte only if the restriction site has beensuccessfully replicated by the DUO extension process.

FIG. 24 shows that the adapter modified analyte (MONO, SEQ ID NOs: 29-35connected as shown in FIG. 18) in the absence of polymerase does notdigest with the restriction enzymes (see gel on the left, Key: M=MfeI,A=AgeI, X=XmaI, N=NgoMIV, B==BspEI), due to the fact they are mismatchedto one another. However, on incubation with polymerase there is anoticeable size shift and the shifted product (DUO, SEQ ID NOs: 29-36connected as shown in FIG. 25) now digests as expected with each of therestriction enzymes (sec gel on the right, Key: M=MfeI, A=AgeI, X=XmaI,N=NgoMIV, B=BspEI).

FIG. 25 shows the polynucleotide DUO hairpin construct (SEQ ID NOs: 29to 36) used in Examples 6.

FIG. 26 shows two typical helicase controlled DNA movements for the DUOhairpin construct (SEQ ID NOs: 29 to 36 connected as shown in FIG. 25)through an MspA nanopore (MS(B1-G75S-G77S-L88N-Q126R)8 MspA (SEQ ID NO:2 with the mutations G75S/G77S1L88N/Q126R)). Sense original region 1.Anti-sense original=region 2. Sense replicate=region 3. Anti-sensereplicate=region 4.

FIG. 27 shows an expanded view of a typical helicase controlled DNAmovement for the DUO hairpin construct (SEQ ID NOs: 29 to 36 connectedas shown in FIG. 25) through an MspA nanopore(MS(B1-G75S-G77S-L88N-Q126R)8 MspA (SEQ ID NO: 2 with the mutationsG75S/G77S/L88N/Q126R)). Sense original=region 1. Anti-senseoriginal=region 2. Sense replicate=region 3. Anti-sense replicate=region4.

FIG. 28 shows an expanded view of a typical transition between the senseoriginal and antisense original regions of the DUO hairpin construct(SEQ ID NOs: 29 to 36 connected as shown in FIG. 25) when under helicasecontrolled DNA movement through an MspA nanopore(MS(B1-G75S-G77S-L88N-Q126R)8 MspA (SEQ ID NO: 2 with the mutationsG75S/G77S/L88N/Q126R)). Sense original=region 1. Anti-senseoriginal=region 2.

DESCRIPTION OF THE SEQUENCE LISTING

SEQ ID NO: 1 shows the codon optimised polynucleotide sequence encodingthe NNN-RRK mutant MspA monomer.

SEQ ID NO: 2 (also referred to as “B1”) shows the amino acid sequence ofthe mature form of the NNN-RRK mutant of the MspA mono mer. The mutantlacks the signal sequence and includes the following mutations: D90N,D91N, D93N, D118R, D134R and E139K. These mutations allow DNA transitionthrough the MspA pore.

SEQ ID NO: 3 shows the polynucleotide sequence encoding one subunit ofα-hemolysin-E111N/K147N (α-HL-NN; Stoddart et al., PNAS, 2009; 106(19):7702-7707).

SEQ ID NO: 4 shows the amino acid sequence of one subunit of α-HL-NN.

SEQ ID NO: 5 shows the codon optimized polynucleotide sequence encodingthe Phi29 DNA polymerase.

SEQ ID NO: 6 shows the amino acid sequence of the Phi29 DNA polymerase.

SEQ ID NOs: 7 to 9 show the amino acid sequences of the mature forms ofthe MspB, C and D mutants respectively. The mature forms lack the signalsequence.

SEQ ID NOs.: 10 to 15 show the sequences used to illustrate homopolymerreads.

SEQ ID NOs: 16 to 36 show the sequences used in the Examples.

DETAILED DESCRIPTION OF THE INVENTION

It is to be understood that different applications of the disclosedproducts and methods may be tailored to the specific needs in the art.It is also to be understood that the terminology used herein is for thepurpose of describing particular embodiments of the invention only, andis not intended to be limiting.

In addition as used in this specification and the appended claims, thesingular forms “a”, “an”, and “the” include plural referents unless thecontent clearly dictates otherwise. Thus, for example, reference to “apore” includes two or more such pores, reference to “a nucleic acidsequence” includes two or more such sequences, and the like.

All publications, patents and patent applications cited herein, whethersupra or infra, are hereby incorporated by reference in their entirety.

Methods of the Invention

The invention provides a method for sequencing a double stranded targetpolynucleotide. The method comprises linking the two strands of thetarget polynucleotide by a bridging moiety. The two strands of thetarget polynucleotide are separated to provide a single strandedpolynucleotide by contacting the construct comprising the targetpolynucleotide with a polynucleotide binding protein. The singlestranded polynucleotide is moved through a transmembrane pore. Aproportion of the nucleotides in the single stranded polynucleotideinteract with the pore. The current passing through the pore is measuredduring each interaction and the sequence of the target polynucleotide isestimated or determined. The target polynucleotide is thereforesequenced using Strand Sequencing. This method may be referred to hereinas the “MONO” method.

As discussed above, linking the two strands of the target polynucleotideby a bridging moiety allows both strands of the target polynucleotide tobe sequenced by the transmembrane pore. This method is advantageousbecause it doubles the amount of information obtained from a singledouble stranded target polynucleotide construct. Moreover, because thesequence in the complementary ‘anti-sense’ strand is necessarilyorthogonal to the sequence of the ‘sense’ strand, the information fromthe two strands can be combined informatically. Thus, this mechanismprovides an orthogonal proof-reading capability that provides higherconfidence observations.

Furthermore, the other major advantages of the method of the inventionare:

1) Coverage of missed nucleotides: the method substantially minimisesissues of any missed nucleotides or groups of nucleotides(e.g. due tomovement issues such as the strand slipping through the pore), since anystates that might be missed in one strand are likely to be covered bythe orthogonal information obtained from its complement region.

2) Coverage of problematic sequence motifs: any difficult to sequencemotifs are covered by the orthogonal and opposite information in thecomplementary strand, which having a different sequence will not havethe same sequence dependent issues. For example, this is particularlyrelevant for sequence motifs that produce only small changes in current,or have similar current levels—i.e. consecutive base motifs that whenmoved through the nanopore produce the same current block, and aretherefore not observed as there is no step change in current. Anysimilar current levels from one sequence motif will be covered by theentirely different current levels obtained from its orthogonal sequencein the complement strand.

In addition to the advantages discussed above there are a number ofspecial cases where the concept of reading both strands of the doublestranded polynucleotide can be utilized to provide further benefits.

1. Epigenetic Information

Being able to identify epigenetic information (such as 5-methylcytosineof 5-hydroxymenthylcytosine nucleotides) or damaged bases within anatural DNA strand is desirable in a wide range of applications. Atpresent, it is difficult to extract this information as the majority ofDNA sequencing technologies rely on DNA amplification as part of theirsequencing chemistry. This information can be extracted, but requireschemical treatment followed by amplification, both of which canintroduce errors.

Nanopore sequencing is also a single molecule sequencing technology andtherefore can be performed without the need of DNA amplification. It hasbeen shown that nanopores can detect modifications to the standard fourDNA nucleotides. Reading both strands of the polynucleotide can beuseful in detecting DNA modifications in situations where a modifiedbase behaves in a similar way (generates a similar current signal) toanother base. For example if methylcytosine (mC) behaves in a similarway to thymidine there is an error associated with assigning a mC to aT. In the sense strand, there is a probability of the base being calleda mC or a T. However, in the anti-sense strand, the corresponding basemay appear as a G with a high probability. Thus by “proofreading” theanti-sense strand, it is highly likely that the base in the sense strandwas a mC rather than a T.

Reading the sense and the anti-sense strand can be performed without theneed of amplification or replication. However, amplification orreplication may be added as part of the sample preparation to aid thedetection of epigenetic information. A nucleotide strand may beconstructed (described in detail below) where the following informationis read through the nanopore in the following order: sense (original),antisense (original), -bridging moiety-, sense (complement), antisense(complement) (FIG. 15).

In this scheme, information on the methylated base will be obtained fourtimes. If the epigenetic base is in the original sense strand (in thiscase, mC), the following information will be obtained with a highprobability: sense (original)-mC, anti-sense (original)-G, sense(complement)-C, and anti-sense (compliment)-G. It is clear that theoriginal sense read and the replicated sense read will give differentresults, while the both anti-sense reads will yield the same base call.This information can be used to indicate the position of the epigeneticbase in the original sense strand.

2. RNA-DNA Double Reads

A similar scheme can be used to sequence RNA. A bridging moiety can beattached to a piece of RNA and the DNA reverse complement added to theRNA via a reverse transcriptase (resulting construct shown in FIG. 16)

In this scheme, the RNA is read followed by a DNA read of the reversecomplement. Information from both the RNA and the DNA reads can becombined to increase the accuracy of determining or estimating the RNAsequence. For example, if a uracil base (U) in RNA gives a similar readto a cytosine, then the corresponding base could be used to resolve thiserror. If the corresponding DNA base is G, then it is highly likely thatthe RNA base was a C, however if the DNA base is called as an A, then itis likely that the RNA base was a U.

3. Homopolymer Reads

Homopolymer reads may be a problem for single molecule nanoporesequencing. If the homopolymer region is longer than the reading sectionof the pore, the length of the homopolymer section will be difficult todetermine.

It has already been shown that Phi29 can be used to read around ahairpin, allowing the sense and the antisense strand to be read.Amplification can be used to generate the antisense strand using apolymerase and a set of regular DNA triphosphates; dTTP, dGTP, dATP,dCTP. To overcome the problem of homopolymner reads, the antisensestrand can be synthesised with the addition of a different base incombination with the original dTTP, dGTP, dATP, dCTP. This could be anatural base analogue such as inosine (I). The base will have a randomchance of incorporating compared to the correct natural base and theinsertion rates can be controlled by varying the concentration of thetriphosphate species.

Through the addition of the alternative base, there will be aprobability of an alternative base being inserted into the reversecomplement of a homopolymer region. The result of this is that thehomopolymer run will be reduced in length to a point where it can beread by the reading section of the nanopore. For example, a homopolymergroup of AAAAAAAAAAAA (SEQ ID NO: 10) will have random insertions of thealternative base and may give TTTITTIITTTI (SEQ ID NO: 11) (where I isinosine).

Original DNA + Hairpin (SEQ ID NO: 12):5′-TTTTTTTTTTTTTTTTTTTTXXXXXTGTACTGCCGTACGTAAAAAAATAGCTGATCGTACTTACTAGCATGTT (abasic-X)Regular Conversion (SEQ TD NO: 13):5′-TTTTTTTTTTTTTTTTTTTTXXXXXTGTACTGCCGTACGTAAAAAAATAGCTGATCGTACTTACATGACGGCATGCATTTTTTATCGACTAGCATGT T (abasic = X)Proposed Scheme 1 (G, T, A, C is randomly replacedby analogue I) (SEQ ID NO 14):5′-TTTTTTTTTTTTTTTTTTTTXXXXXTGTACTGCCGTACGTAAAAAAATAGCTGATCGTACTTAIATIACGICATGIATTITTITATIGACTAGCATG TT (abasic = X)The base analogue could be generic (replace T, G, A, or C), or it couldbe specific to one base (e.g. deoxyuridine (U) just replaces T).

Proposed Scheme 2 (T is randomly replaced by analogue U) (SEQ ID NO: 15):5′-TTTTTTTTTTTTTTTTTTTTXXXXXTGTACTGCCGTACGTAAAAAAATAGCTGATCGTACTTACAUGACGGCATGCAUTTTUTTATCGACTAGCATG TT (abasic = X)

In both scheme one and two, the homopolymer stretch has been reduced toallow individual nucleotides or groups of nucleotides to be estimated ordetermined. The sense strand will be a natural DNA strand, while theanti-sense will contain a mixture of natural bases and base analogues.The combination of data from the sense and the antisense reads can beused to estimate the length of the homopolymer run in the original DNAsection.

Double Stranded Target Polynucleotide

The method of the invention is for sequencing a double strandedpolynucleotide. A polynucleotide, such as a nucleic acid, is amacromolecule comprising two or more nucleotides. The polynucleotide ornucleic acid may comprise any combination of any nucleotides. Thenucleotides can be naturally occurring or artificial. The nucleotide canbe oxidized or methylated. A nucleotide typically contains a nucleobase,a sugar and at least one phosphate group. The nucleobase is typicallyheterocyclic. Nucleobases include, but are not limited to, purines andpyrimidines and more specifically adenine, guanine, thymine, uracil andcytosine. The sugar is typically a pentose sugar. Nucleotide sugarsinclude, but are not limited to, ribose and deoxyribose. The nucleotideis typically a ribonucleotide or deoxyribonucleotide. The nucleotidetypically contains a monophosphate, diphosphate or triphosphate.Phosphates may be attached on the 5′ or 3′ side of a nucleotide.

Nucleotides include, but are not limited to, adenosine monophosphate(AMP), adenosine diphosphate (ADP), adenosine triphosphate (ATP),guanosine monophosphate (GMP), guanosine diphosphate (GDP), guanosinetriphosphate (GTP), thymidine monophosphate (TMP), thymidine diphosphate(TDP), thymidine triphosphate (TTP), uridine monophosphate (UMP),uridine diphosphate (UDP), uridine triphosphate (UTP), cytidinemonophosphate (CMP), cytidine diphosphate (CDP), cytidine triphosphate(CTP), 5-methylcytidine monophosphate, 5-methylcytidine diphosphate,5-methylcytidine triphosphate, 5-hydroxymethylcytidine monophosphate,5-hydroxymethylcytidine diphosphate, 5-hydroxymethylcytidinetriphosphate, cyclic adenosine monophosphate (cAMP), cyclic guanosinemonophosphate (cGMP), deoxyadenosine monophosphate (dAMP),deoxyadenosine diphosphate (dADP), deoxyadenosine triphosphate (dATP),deoxyguanosine monophosphate (dGMP), deoxyguanosine diphosphate (dGDP),deoxyguanosine triphosphate (dGTP), deoxythymidine monophosphate (dTMP),deoxythymidine diphosphate (dTDP), deoxythymidine triphosphate (dTTP),deoxyuridine monophosphate (dUMP), deoxy uridine diphosphate (dUDP),deoxyuridine triphosphate (dUTP), deoxycytidine monophosphate (dCMP),deoxycytidine diphosphate (dCDP) and deoxycytidine triphosphate (dCTP),5-methyl-2′-deoxycytidine monophosphate, 5-methyl-2′-deoxycytidinediphosphate, 5-methyl-2′-deoxycytidine triphosphate,5-hydroxymethyl-2′-deoxycytidine monophosphate,5-hydroxymethyl-2′-deoxycytidine diphosphate and5-hydroxymethyl-2′-deoxycytidine triphosphate. The nucleotides arepreferably selected from AMP, TMP, GMP, CMP, UMP, dAMP, dTMP, dGMP ordCMP.

A nucleotide may contain a sugar and at least one phosphate group (i.e.lack a nucleobase).

The polynucleotide can be a nucleic acid, such as deoxyribonucleic acid(DNA) or ribonucleic acid (RNA). The target polynucleotide can compriseone strand of RNA hybridized to one strand of DNA. The polynucleotidemay be any synthetic nucleic acid known in the art, such as peptidenucleic acid (PNA), glycerol nucleic acid (GNA), threose nucleic acid(TNA), locked nucleic acid (LNA) or other synthetic polymers withnucleotide side chains.

The target polynucleotide can be any length. For example, thepolynucleotide can be at least 10, at least 50, at least 100, at least150, at least 200, at least 250, at least 300, at least 400 or at least500 nucleotide pairs in length. The polynucleotide can be 1000 or morenucleotide pairs, 5000 or more nucleotide pairs in length or 100000 ormore nucleotide pairs in length.

The target polynucleotide is present in any suitable sample. Theinvention is typically carried out on a sample that is known to containor suspected to contain the target polynucleotide. Alternatively, theinvention may be carried out on a sample to confirm the identity of oneor more target polynucleotides whose presence in the sample is known orexpected.

The sample may be a biological sample. The invention may be carried outin vitro on a sample obtained from or extracted from any organism ormicroorganism. The organism or microorganism is typically archean,prokaryotic or eukaryotic and typically belongs to one the fivekingdoms: plantae, animalia, fungi, monera and protista. The inventionmay be carried out in vitro on a sample obtained from or extracted fromany virus. The sample is preferably a fluid sample. The sample typicallycomprises a body fluid of the patient. The sample may be urine, lymph,saliva, mucus or amniotic fluid but is preferably blood, plasma orserum. Typically, the sample is human in origin, but alternatively itmay be from another mammal animal such as from commercially farmedanimals such as horses, cattle, sheep or pigs or may alternatively bepets such as cats or dogs. Alternatively a sample of plant origin istypically obtained from a commercial crop, such as a cereal, legume,fruit or vegetable, for example wheat, barley, oats, canola, maize,soya, rice, bananas, apples, tomatoes, potatoes, grapes, tobacco, beans,lentils, sugar cane, cocoa, cotton, tea, coffee.

The sample may be a non-biological sample. The non-biological sample ispreferably a fluid sample. Examples of a non-biological sample includesurgical fluids, water such as drinking water, sea water or river water,and reagents for laboratory tests.

The sample is typically processed prior to being assayed, for example bycentrifugation or by passage through a membrane that filters outunwanted molecules or cells, such as red blood cells. The sample may bemeasured immediately upon being taken. The sample may also be typicallystored prior to assay, preferably below −70° C.

If the target polynucleotide is coupled to the membrane as discussed inmore detail below, the method of the invention is particularlyadvantageous for human DNA sequencing because only small amounts ofpurified DNA can be obtained from human blood. The method preferablyallows sequencing of a target polynucleotide that is present at aconcentration of from about 0.1 pM to about 1 nM, such as less than 1pM, less than 10 pM or less than 100 pM.

Construct

The method of the invention involves providing a construct comprisingthe double stranded target nucleotide to be sequenced. The constructtypically allows both strands of the target polynucleotide to besequenced by a transmembrane pore.

The construct comprises a bridging moiety which is capable of linkingthe two strands of the target polynucleotide. The bridging moietytypically covalently links the two strands of the target polynucleotide.The bridging moiety can be anything that is capable of linking the twostrands of the target polynucleotide, provided that the bridging moietydoes not interfere with movement of the single stranded polynucleotidethrough the transmembrane pore. Suitable bridging moieties include, butare not limited to a polymeric linker, a chemical linker, apolynucleotide or a polypeptide. Preferably, the bridging moietycomprises DNA, RNA, modified DNA (such as abasic DNA), RNA, PNA, LNA orPEG. The bridging moiety is more preferably DNA or RNA.

The bridging moiety is most preferably a hairpin loop. The hairpin loopis typically 4 to 100 nucleotides in length, preferably 4 to 8nucleotides in length.

The bridging moiety is linked to the target polynucleotide construct byany suitable means known in the art. The bridging moiety may besynthesized separately and chemically attached or enzymatically ligatedto the target polynucleotide. Alternatively, the bridging moiety may begenerated in the processing of the target polynucleotide.

The bridging moiety is linked to the target polynucleotide at or nearone end of the target polynucleotide. The bridging moiety is preferablylinked to the target polynucleotide within 10 nucleotides of the end ofthe target polynucleotide.

The construct comprising the target polynucleotide also preferablycomprises at least one polymer at the opposite end of the targetpolynucleotide to the bridging moiety. Such polymer(s) aid thesequencing method of the invention as discussed in more detail below.Suitable polymers include polynucleotides (DNA/RNA), modifiedpolynucleotides such as modified DNA, PNA, LNA, PEG or polypeptides.

The construct preferably comprises a leader polymer. The leader polymeris linked to the target polynucleotide at the opposite end to thebridging moiety. The leader polymer helps the double stranded targetpolynucleotide to engage with the transmembrane pore or with apolynucleotide binding protein, such as Phi29 DNA polymerase, that helpsto separate the two strands and/or controls the movement of the singlestranded polynucleotide through the pore. Transmembrane pores andpolynucleotide binding proteins are discussed in more detail below.

The leader polymer can be a polynucleotide such as DNA or RNA, amodified polynucleotide (such as abasic DNA), PNA, LNA, PEG or apolypeptide. The leader polymer is preferably a polynucleotide and ismore preferably a single stranded polynucleotide. The leader polymer canbe any of the polynucleotides discussed above. The single strandedleader polymer is most preferably a single strand of DNA. The leaderpolymer can be any length, but is typically 27 to 150 nucleotides inlength, such as from 50 to 150 nucleotides in length.

The addition of sections of single stranded polynucleotide to a doublestranded polynucleotide can be performed in various ways. A chemical orenzymatic ligation can be done. In addition, the Nextera method byEpicentre is suitable. The inventors have developed a PCR method using asense primer that, as usual contains a complementary section to thestart of the target region of genomic DNA, but was additionally precededwith a 50 polyT section. To prevent the polymerase from extending thecomplementary strand opposite the polyT section and thereby create ablunt ended PCR product (as is normal), four abasic sites were addedbetween the polyT section and the complimentary priming section. Theseabasic sites will prevent the polymerase from extending beyond thisregion and so the polyT section will remain as 5′ single stranded DNA oneach of the amplified copies. Other possible modifications which couldalso stop polymerase extension include RNA, PNA or morpholino bases,iso-dC or iso-dG.

The construct preferably further comprises a polymer tail (also linkedto the target polynucleotide at the opposite end to the bridgingmoiety). The polymer tail aids sequencing of the target construct by thetransmembrane pore. In particular, the polymer tail typically ensuresthat the entirety of the double stranded polynucleotide (i.e. all ofboth strands) can be read and sequenced by the transmembrane pore. Asdiscussed below, polynucleotide binding proteins, such as Phi29 DNApolymerase, can control the movement of the single strandedpolynucleotides through the transmembrane pore. The protein typicallyslows the movement of the polynucleotide through the pore. For instance,Phi29 DNA polymerase acts like a brake slowing the movement of thepolynucleotide through the pore along the potential applied across themembrane. Once the polynucleotide is no longer in contact with thebinding protein, it is free to move through the pore at such a rate thatsequence information is difficult to obtain. Since there is normally ashort distance from the protein to the pore, typically approximately 20nucleotides some sequence information (approximately equal to thatdistance) may be missed. A tail polymer “extends” the length of thesingle stranded polynucleotide such that its movement may be controlledby the nucleic acid binding protein while all of both strands of thetarget polynucleotide pass through the pore and are sequenced. Suchembodiments ensure that sequence information can be obtained from theentirety of both strands in the target polynucleotide. The tail polymermay also provide a site for a primer to bind, which allows the nucleicacid binding protein to separate the two strands of the targetpolynucleotide.

The tail polymer can be a polynucleotide such as DNA or RNA, a modifiedpolynucleotide (such as abasic DNA), PNA, LNA, PEG or a polypeptide. Thetail polymer is preferably a polynucleotide and is more preferably asingle stranded polynucleotide. The tail polymer can be any of thepolynucleotides discussed above.

The construct preferably also comprises one or more markers, whichresult in a distinctive current (characteristic signature current) whenpassed through the transmembrane pore. The markers are typically used toallow the position of the single stranded polynucleotide in relation tothe pore to be estimated or determined. For instance, the signal from amarker positioned between both strands of the target polynucleotideindicates that one strand has been sequenced and the other is about toenter the pore. Hence, such markers can be used to differentiate betweenthe sense and anti-sense strands of target DNA. The marker(s) may alsobe used to identify the source of the target polynucleotide. Suitablemarkers include, but are not limited to abasic regions, specificsequences of nucleotides, unnatural nucleotides, fluorophores orcholesterol. The markers are preferably an abasic region or a specificsequence of nucleotides.

The marker(s) may be positioned anywhere in the construct. The marker(s)can be positioned in the bridging moiety. The marker(s) can also bepositioned near the bridging moiety. Near the bridging moiety preferablyrefers to within 10 to 100 nucleotides of the bridging moiety.

The markers can also be positioned within the leader polymer or the tailpolymer.

The construct may be coupled to the membrane using any known method. Ifthe membrane is an amphiphilic layer, such as a lipid bilayer (asdiscussed in detail below), the construct is preferably coupled to themembrane via a polypeptide present in the membrane or a hydrophobicanchor present in the membrane. The hydrophobic anchor is preferably alipid, fatty acid, sterol, carbon nanotube or amino acid.

The construct may be coupled directly to the membrane. The construct ispreferably coupled to the membrane via a linker. Preferred linkersinclude, but are not limited to, polymers, such as polynucleotides,polyethylene glycols (PEGs) and polypeptides. If a polynucleotide iscoupled directly to the membrane, then some sequence data will be lostas the sequencing run cannot continue to the end of the polynucleotidedue to the distance between the membrane and the detector. If a linkeris used, then the polynucleotide can be processed to completion. If alinker is used, the linker may be attached to the construct at anyposition. The linker is preferably attached to the polynucleotide at thetail polymer.

The coupling may be stable or transient. For certain applications, thetransient nature of the coupling is preferred. If a stable couplingmolecule were attached directly to either the 5′ or 3′ end of apolynucleotide, then some sequence data will be lost as the sequencingrun cannot continue to the end of the polynucleotide due to the distancebetween the bilayer and the enzymes active site. If the coupling istransient, then when the coupled end randomly becomes free of thebilayer, then the polynucleotide can be processed to completion.Chemical groups that form stable or transient links with the membraneare discussed in more detail below. The construct may be transientlycoupled to an amphiphilic layer or lipid bilayer using cholesterol or afatty acyl chain. Any fatty acyl chain having a length of from 6 to 30carbon atoms, such as hexadecanoic acid, may be used.

In preferred embodiments, construct is coupled to an amphiphilic layersuch as a lipid bilayer. Coupling of polynucleotides to synthetic lipidbilayers has been carried out previously with various differenttethering strategies. These are summarised in Table 1 below.

TABLE 1 Attach- ment Type of group coupling Reference Thiol StableYoshina-Ishii, C. and S. G. Boxer (2003). “Arrays of mobile tetheredvesicles on supported lipid bilayers.” J Am Chem Soc 125(13): 3696-7.Biotin Stable Nikolov, V., R. Lipowsky, et al. (2007). “Behavior ofgiant vesicles with anchored DNA molecules.” Biophys J 92(12): 4356-68Choles- Transient Pfeiffer, I. and F. Hook (2004). “Bivalent terolcholesterol-based coupling of oligonucletides to lipid membraneassemblies.” J Am Chem Soc 126(33): 10224-5 Lipid Stable van Lengerich,B., R. J. Rawle, et al. “Covalent attachment of lipid vesicles to afluid-supported bilayer allows observation of DNA-mediated vesicleinteractions.” Langmuir 26(11): 8666-72

Polynucleotides may be functionalized using a modified phosphoramiditein the synthesis reaction, which is easily compatible for the additionof reactive groups, such as thiol, cholesterol, lipid and biotin groups.These different attachment chemistries give a suite of attachmentoptions for polynucleotides. Each different modification group tethersthe polynucleotide in a slightly different way and coupling is notalways permanent so giving different dwell times for the polynucleotideto the bilayer. The advantages of transient coupling are discussedabove.

Coupling of polynucleotides can also be achieved by a number of othermeans provided that a reactive group can be added to the polynucleotide.The addition of reactive groups to either end of DNA has been reportedpreviously. A thiol group can be added to the 5′ of ssDNA usingpolynucleotide kinase and ATPγS (Grant, G. P. and P. Z. Qin (2007). “Afacile method for attaching nitroxide spin labels at the 5′ terminus ofnucleic acids.” Nucleic Acids Res 35(10): e77). A more diverse selectionof chemical groups, such as biotin, thiols and fluorophores, can beadded using terminal transferase to incorporate modifiedoligonucleotides to the 3′ of ssDNA (Kumar, A., P. Tehen, et al. (1988).“Nonradioactive labeling of synthetic oligonucleotide probes withterminal deoxynucleotidyl transferase.” Anal Biochem 169(2): 376-82).

Alternatively, the reactive group could be considered to be the additionof a short piece of DNA complementary to one already coupled to thebilayer, so that attachment can be achieved via hybridisation. Ligationof short pieces of ssDNA have been reported using T4 RNA ligase I(Troutt, A. B., M. G. McHeyzer-Williams, et al. (1992).“Ligation-anchored PCR: a simple amplification technique withsingle-sided specificity.” Proc Natl Acad Sci USA 89(20): 9823-5).Alternatively either ssDNA or dsDNA could be ligated to native dsDNA andthen the two strands separated by thermal or chemical denaturation. Tonative dsDNA, it is possible to add either a piece of ssDNA to one orboth of the ends of the duplex, or dsDNA to one or both ends. Then, whenthe duplex is melted, each single strand will have either a 5′ or 3′modification if ssDNA was used for ligation or a modification at the 5′end, the 3′ end or both if dsDNA was used for ligation. If thepolynucleotide is a synthetic strand, the coupling chemistry can beincorporated during the chemical synthesis of the polynucleotide. Forinstance, the polynucleotide can be synthesized using a primer with areactive group attached to it.

A common technique for the amplification of sections of genomic DNA isusing polymerase chain reaction (PCR). Here, using two syntheticoligonucleotide primers, a number of copies of the same section of DNAcan be generated, where for each copy the 5′ of each strand in theduplex will be a synthetic polynucleotide. By using an antisense primerthat has a reactive group, such as a cholesterol, thiol, biotin orlipid, each copy of the target DNA amplified will contain a reactivegroup for coupling.

Separating

The two strands of the target polynucleotide are separated using apolynucleotide binding protein.

The polynucleotide binding protein is preferably derived from apolynucleotide handling enzyme. However, the enzyme may be used underconditions in which is does not catalyze a reaction. For instance, aprotein derived from Phi29 DNA polymerase may be run in an unzippingmode as discussed in more detail below.

A polynucleotide handling enzyme is a polypeptide that is capable ofinteracting with and modifying at least one property of apolynucleotide. The enzyme may modify the polynucleotide by cleaving itto form individual nucleotides or shorter chains of nucleotides, such asdi- or trinucleotides. The enzyme may modify the polynucleotide byorienting it or moving it to a specific position. The polynucleotidehandling enzyme does not need to display enzymatic activity as long asit is capable of binding the target polynucleotide and preferablycontrolling its movement through the pore. For instance, the enzyme maybe modified to remove its enzymatic activity or may be used underconditions which prevent it from acting as an enzyme. Such conditionsare discussed in more detail below.

The polynucleotide binding protein is typically derived from thePicovirinae virus family. Suitable viruses include, but are not limitedto, AHJD-like viruses and Phi29 like viruses. The polynucleotide bindingprotein is preferably derived from Phi29 DNA polymerase or a helicase.

A protein derived from Phi29 DNA polymerase comprises the sequence shownin SEQ ID NO: 6 or a variant thereof. Wild-type Phi29 DNA polymerase haspolymerase and exonuclease activity. It may also unzip double strandedpolynucleotides under the correct conditions. Hence, the enzyme may workin three modes. This is discussed in more detail below. A variant of SEQID NOs: 6 is an enzyme that has an amino acid sequence which varies fromthat of SEQ ID NO: 6 and which retains polynucleotide binding activity.The variant must work in at least one of the three modes discussedbelow. Preferably, the variant works in all three modes. The variant mayinclude modifications that facilitate handling of the polynucleotideand/or facilitate its activity at high salt concentrations and/or roomtemperature. The variant may include Fidelity Systems' TOPOmodification, which improves enzyme salt tolerance.

Over the entire length of the amino acid sequence of SEQ 1D NO: 6, avariant will preferably be at least 40% homologous to that sequencebased on amino acid identity. More preferably, the variant polypeptidemay be at least 50%, at least 55%, at least 60%, at least 65%, at least70%, at least 75%, at least 80%, at least 85%, at least 90% and morepreferably at least 95%, 97% or 99% homologous based on amino acididentity to the amino acid sequence of SEQ ID NO: 6 over the entiresequence. There may be at least 80%, for example at least 85%, 90% or95%, amino acid identity over a stretch of 200 or more, for example 230,250, 270 or 280 or more, contiguous amino acids (“hard homology”).Homology is determined as described below. The variant may differ fromthe wild-type sequence in any of the ways discussed below with referenceto SEQ ID NO: 2. The enzyme may be covalently attached to the pore asdiscussed below.

The method is preferably carried out using the protein derived fromPhi29 DNA polymerase in unzipping mode. In this embodiment, steps (b),(c) and (d) are carried out in the absence of free nucleotides and theabsence of an enzyme cofactor such that the polymerase controls themovement of the single stranded polynucleotide through the pore with thefield resulting from the applied voltage (as it is unzipped). In thisembodiment, the polymerase acts like a brake preventing the singlestranded polynucleotide from moving through the pore too quickly underthe influence of the applied voltage. The method preferably furthercomprises (e) lowering the voltage applied across the pore such that thesingle stranded polynucleotide moves through the pore in the oppositedirection to that in steps (c) and (d) (i.e. as it re-anneals) and aproportion of the nucleotides in the polynucleotide interacts with thepore and (f) measuring the current passing through the pore during eachinteraction and thereby proof reading the sequence of the targetpolynucleotide obtained in step (d), wherein steps (e) and (f) are alsocarried out with a voltage applied across the pore.

The two strands of the target polynucleotide can be separated andduplicated at any stage before sequencing is carried out and as manytimes as necessary. For example, after separating the two strands of afirst target polynucleotide construct as described above, acomplementary strand to the resulting single stranded polynucleotide canbe generated to form another double stranded polynucleotide. The twostrands of this double stranded polynucleotide can then be linked usinga bridging moiety to form a second construct. This may be referred toherein as the “DUO” method. This construct may then be used in theinvention. In such an embodiment, one strand of the double strandedpolynucleotide in the resulting construct contains both strands of theoriginal target double stranded polynucleotide (in the first construct)linked by a bridging moiety. The sequence of the original target doublestranded polynucleotide or the complement strand can be estimated ordetermined. This process of replication can be repeated as many times asnecessary and provides additional proofreading as the targetpolynucleotide is in effect being read multiple times.

Membrane

Any membrane may be used in accordance with the invention. Suitablemembranes are well-known in the art. The membrane is preferably anamphiphilic layer. An amphiphilic layer is a layer formed fromamphiphilic molecules, such as phospholipids, which have bothhydrophilic and lipophilic properties. The amphiphilic molecules may besynthetic or naturally occurring. Non-naturally occurring amphiphilesand amphiphiles which form a monolayer are known in the art and include,for example, block copolymers (Gonzalez-Perez et al., Langmuir, 2009,25, 10447-10450). Block copolymers are polymeric materials in which twoor more monomer sub-units that are polymerized together to create asingle polymer chain. Block copolymers typically have properties thatare contributed by each monomer sub-unit. However, a block copolymer mayhave unique properties that polymers formed from the individualsub-units do not possess. Block copolymers can be engineered such thatone of the monomer sub-units is hydrophobic (i.e. lipophilic), whilstthe other sub-unit(s) are hydrophilic whilst in aqueous media. In thiscase, the block copolymer may possess amphiphilic properties and mayform a structure that mimics a biological membrane. The block copolymermay be a diblock (consisting of two monomer sub-units), but may also beconstructed from more than two monomer sub-units to form more complexarrangements that behave as amphiphiles. The copolymer may be atriblock, tetrablock or pentablock copolymer.

Archaebacterial bipolar tetraether lipids are naturally occurring lipidsthat are constructed such that the lipid forms a monolayer membrane.These lipids are generally found in extremophiles that survive in harshbiological environments, thermopiles, halophiles and acidophiles. Theirstability is believed to derive from the fused nature of the finalbilayer. It is straightforward to construct block copolymer materialsthat mimic these biological entities by creating a triblock polymer thathas the general motif hydrophilic-hydrophobic-hydrophilic. This materialmay form monomeric membranes that behave similarly to lipid bilayers andencompass a range of phase behaviours from vesicles through to laminarmembranes. Membranes formed from these triblock copolymers hold severaladvantages over biological lipid membranes. Because the triblockcopolymer is synthesized, the exact construction can be carefullycontrolled to provide the correct chain lengths and properties requiredto form membranes and to interact with pores and other proteins.

Block copolymers may also be constructed from sub-units that are notclassed as lipid sub-materials; for example a hydrophobic polymer may bemade from siloxane or other non-hydrocarbon based monomers. Thehydrophilic sub-section of block copolymer can also possess low proteinbinding properties, which allows the creation of a membrane that ishighly resistant when exposed to raw biological samples. This head groupunit may also be derived from non-classical lipid head-groups.

Triblock copolymer membranes also have increased mechanical andenvironmental stability compared with biological lipid membranes, forexample a much higher operational temperature or pH range. The syntheticnature of the block copolymers provides a platform to customize polymerbased membranes for a wide range of applications.

The amphiphilic molecules may be chemically-modified or functionalisedto facilitate coupling of the analyte.

The amphiphilic layer may be a monolayer or a bilayer. The amphiphiliclayer is typically planar. The amphiphilic layer may be non-planar suchas curved.

The amphiphilic layer is typically a lipid bilayer. Lipid bilayers aremodels of cell membranes and serve as excellent platforms for a range ofexperimental studies. For example, lipid bilayers can be used for invitro investigation of membrane proteins by single-channel recording.Alternatively, lipid bilayers can be used as biosensors to detect thepresence of a range of substances. The lipid bilayer may be any lipidbilayer. Suitable lipid bilayers include, but are not limited to, aplanar lipid bilayer, a supported bilayer or a liposome. The lipidbilayer is preferably a planar lipid bilayer. Suitable lipid bilayersare disclosed in International Application No. PCT/GB08/000563(published as WO 2008/102121), International Application No.PCT/GB08/004127 (published as WO 2009/077734) and InternationalApplication No. PCT/GB2006/001057 (published as WO 2006/100484).

Methods for forming lipid bilayers are known in the art. Suitablemethods are disclosed in the Example. Lipid bilayers are commonly formedby the method of Montal and Mueller (Proc. Natl. Acad. Sci. USA., 1972;69: 3561-3566), in which a lipid monolayer is carried on aqueoussolution/air interface past either side of an aperture which isperpendicular to that interface.

The method of Montal & Mueller is popular because it is a cost-effectiveand relatively straightforward method of forming good quality lipidbilayers that are suitable for protein pore insertion. Other commonmethods of bilayer formation include tip-dipping, painting bilayers andpatch-clamping of liposome bilayers.

In a preferred embodiment, the lipid bilayer is formed as described inInternational Application No. PCT/GB08/004127 (published as WO2009/077734). Advantageously in this method, the lipid bilayer is formedfrom dried lipids. In a most preferred embodiment, the lipid bilayer isformed across an opening as described in WO2009/0777734(PCT/GB08/004127).

In another preferred embodiment, the membrane is a solid state layer. Asolid-state layer is not of biological origin. In other words, a solidstate layer is not derived from or isolated from a biologicalenvironment such as an organism or cell, or a synthetically manufacturedversion of a biologically available structure. Solid state layers can beformed from both organic and inorganic materials including, but notlimited to, microelectronic materials, insulating materials such asSi3N4, Al203, and SiO, organic and inorganic polymers such as polyamide,plastics such as Teflon® or elastomers such as two-componentaddition-cure silicone rubber, and glasses. The solid state layer may beformed from graphene. Suitable graphene layers are disclosed inInternational Application No. PCT/US2008/010637 (published as WO2009/035647).

Transmembrane Pore

A transmembrane pore is a structure that permits hydrated ions driven byan applied potential to flow from one side of the membrane to the otherside of the membrane.

The transmembrane pore is preferably a transmembrane protein pore. Atransmembrane protein pore is a polypeptide or a collection ofpolypeptides that permits hydrated ions, such as analyte, to flow fromone side of a membrane to the other side of the membrane. In the presentinvention, the transmembrane protein pore is capable of forming a porethat permits hydrated ions driven by an applied potential to flow fromone side of the membrane to the other. The transmembrane protein porepreferably permits analyte such as nucleotides to flow from one side ofthe membrane, such as a lipid bilayer, to the other. The transmembraneprotein pore allows a polynucleotide, such as DNA or RNA, to be movedthrough the pore.

The transmembrane protein pore may be a monomer or an oligomer. The poreis preferably made up of several repeating subunits, such as 6, 7 or 8subunits. The pore is more preferably a heptameric or octameric pore.

The transmembrane protein pore typically comprises a barrel or channelthrough which the ions may flow. The subunits of the pore typicallysurround a central axis and contribute strands to a transmembrane 3barrel or channel or a transmembrane α-helix bundle or channel.

The barrel or channel of the transmembrane protein pore typicallycomprises amino acids that facilitate interaction with analyte, such asnucleotides, polynucleotides or nucleic acids. These amino acids arepreferably located near a constriction of the barrel or channel. Thetransmembrane protein pore typically comprises one or more positivelycharged amino acids, such as arginine, lysine or histidine, or aromaticamino acids, such as tyrosine or tryptophan. These amino acids typicallyfacilitate the interaction between the pore and nucleotides,polynucleotides or nucleic acids.

Transmembrane protein pores for use in accordance with the invention canbe derived from β-barrel pores or α-helix bundle pores. β-barrel porescomprise a barrel or channel that is formed from β-strands. Suitableβ-barrel pores include, but are not limited to, β-toxins, such asα-hemolysin, anthrax toxin and leukocidins, and outer membraneproteins/porins of bacteria, such as, Mycobacterium smegmatis porin(Msp), for example MspA, outer membrane porin F (OmpF), outer membraneporin G (OmpG), outer membrane phospholipase A and Neisseriaautotransporter lipoprotein (NalP). α-helix bundle pores comprise abarrel or channel that is formed from α-helices. Suitable α-helix bundlepores include, but are not limited to, inner membrane proteins and αouter membrane proteins, such as WZA and ClyA toxin. The transmembranepore may be derived from Msp or from α-hemolysin (α-HL).

The transmembrane protein pore is preferably derived from Msp,preferably from MspA. Such a pore will be oligomeric and typicallycomprises 7, 8, 9 or 10 monomers derived from Msp. The pore may be ahomo-oligomeric pore derived from Msp comprising identical monomers.Alternatively, the pore may be a hetero-oligomeric pore derived from Mspcomprising at least one monomer that differs from the others. The poremay also comprise one or more constructs which comprise two or morecovalently attached monomers derived from Msp. Suitable pores aredisclosed in U.S. Provisional Application No. 61/441,718 (filed 11 Feb.2011). Preferably the pore is derived from MspA or a homolog or paralogthereof.

A monomer derived from Msp comprises the sequence shown in SEQ ID NO: 2or a variant thereof. SEQ ID NO: 2 is the NNN-RRK mutant of the MspAmonomer. It includes the following mutations: D90N, D91N, D93N, D118R,D134R and E139K. A variant of SEQ ID NO: 2 is a polypeptide that has anamino acid sequence which varies from that of SEQ ID NO: 2 and whichretains its ability to form a pore. The ability of a variant to form apore can be assayed using any method known in the art. For instance, thevariant may be inserted into a lipid bilayer along with otherappropriate subunits and its ability to oligomerise to form a pore maybe determined. Methods are known in the art for inserting subunits intomembranes, such as lipid bilayers. For example, subunits may besuspended in a purified form in a solution containing a lipid bilayersuch that it diffuses to the lipid bilayer and is inserted by binding tothe lipid bilayer and assembling into a fictional state. Alternatively,subunits may be directly inserted into the membrane using the “pick andplace” method described in M. A. Holden, H. Bayley. J. Am. Chem. Soc.2005, 127, 6502-6503 and International Application No. PCT/GB2006/001057(published as WO 2006/100484).

Preferred variants are disclosed in International Application No.PCT/GB2012/050301 (claiming priority from US Provisional Application No.61/441,718). Particularly preferred variants include, but are notlimited to, those comprising the following substitution(s): L88N; L88S;L88Q; L88T; D90S; D90Q; D90Y; I105L; I105S; Q126R; G75S; G77S; G75S,077S, L88N and Q126R; G75S, G77S, L88N, D90Q and Q126R; D90Q and Q126R;L88N, D90Q and Q126R; L88S and D90Q; L88N and D90Q; E59R; G75Q; G75N;G75S; G75T; G77Q; G77N; G77S; G77T; I78L; S81N; T83N; N86S; N86T; I87F;I87V; I87L; L88N; L88S; L88Y; L88F; L88V; L88Q; L88T; I89F; I89V; I89L;N90S; N90Q; N90L; N90Y; N91S; N91Q; N91L; N91M; N91I; N91A; N91V; N91G;G92A; G92S; N93S; N93A; N93T; I94L; T95V; A96R; A96D; A96V; A96N; A96S;A96T; P97S; P98S; F99S; G100S; L101F; N102K; N102S; N102T; S103A; S103Q;S103N; S103G; S103T: V104I; I105Y; I105L; I105A; I105G; I105Q; I105N;I105S; I105T; T106F; T106I; T106V; T106S; N108P; N108S; D90Q and I105A;D90S and G92S; L88T and D90S; I87Q and D90S; I89Y and D90S: L88N andI89F; L88N and I89Y; D90S and G92A; D90S and I94N; D90S and V104I; L88Dand I105K; L88N and Q126R; L88N, D90Q and D91R; L88N, D90Q and D91S;L88N, D90Q and I105V; D90Q, D93S and I105A; N91 Y; N90Y and N91G; N900and N91 Y; N90G and N91G; I050; N90R; N91R; N90R and N91R; N90K; N91K;N90K and N91K; N90Q and N91G; N90G and N91Q; N90Q and N91Q; R118N; N91C;N90C; N90W; N91W; N90K; N91K; N90R; N91R; N90S and N91S; N90Y and I105A;N90G and I105A; N90Q and I105A; N90S and I105A; L88A and I105A; L88S andI105S; L88N and I105N; N90G and N93G; N90G; N93G; N90G and N91A; I105K;I105R; I105V; I105P; I105W; L88R; L88A; L88G; L88N; N90R and I105A; N90Sand I105A; L88A and I105A; L88S and I105S; L88N and I105N; L88C; S103C;I105C; D134R.

In addition to the specific mutations discussed above, the variant mayinclude other mutations. Over the entire length of the amino acidsequence of SEQ ID NO: 2, a variant will preferably be at least 50%homologous to that sequence based on amino acid identity. Morepreferably, the variant may be at least 55%, at least 60%, at least 65%,at least 70%, at least 75%, at least 80%, at least 85%, at least 90% andmore preferably at least 95%, 97% or 99% homologous based on amino acididentity to the amino acid sequence of SEQ ID NO: 2 over the entiresequence. There may be at least 80%, for example at least 85%, 90% or95%, amino acid identity over a stretch of 100 or more, for example 125,150, 175 or 200 or more, contiguous amino acids (“hard homology”).

Standard methods in the art may be used to determine homology. Forexample the UWGCG Package provides the BESTFIT program which can be usedto calculate homology, for example used on its default settings(Devereux et al (1984) Nucleic Acids Research 12, p387-395). The PILEUPand BLAST algorithms can be used to calculate homology or line upsequences (such as identifying equivalent residues or correspondingsequences (typically on their default settings)), for example asdescribed in Altschul S. F. (1993) J Mol Evol 36:290-300; Altschui, S. Fet al (1990) J Mol Biol 215:403-10. Software for performing BLASTanalyses is publicly available through the National Center forBiotechnology Information (http://www.ncbi.nlm.nih.gov/).

SEQ ID NO: 2 is the NNN-RRK mutant of the MspA monomer. The variant maycomprise any of the mutations in the MspB, C or D monomers compared withMspA. The mature forms of MspB, C and D are shown in SEQ ID NOs: 7 to 9.In particular, the variant may comprise the following substitutionpresent in MspB: A138P. The variant may comprise one or more of thefollowing substitutions present in MspC: A96G, N102E and A138P. Thevariant may comprise one or more of the following mutations present inMspD: Deletion of G1, L2V, E5Q, L8V, D13G, W21A, D22E, K47T, I49H, I68V,D91G, A96Q, N102D, S103T, V104I, S136K and G141A. The variant maycomprise combinations of one or more of the mutations and substitutionsfrom Msp B. C and D.

Amino acid substitutions may be made to the amino acid sequence of SEQID NO: 2 in addition to those discussed above, for example up to 1, 2,3, 4, 5, 10, 20 or 30 substitutions. Conservative substitutions replaceamino acids with other amino acids of similar chemical structure,similar chemical properties or similar side-chain volume. The aminoacids introduced may have similar polarity, hydrophilicity,hydrophobicity, basicity, acidity, neutrality or charge to the aminoacids they replace. Alternatively, the conservative substitution mayintroduce another amino acid that is aromatic or aliphatic in the placeof a pre-existing aromatic or aliphatic amino acid. Conservative aminoacid changes are well-known in the art and may be selected in accordancewith the properties of the 20 main amino acids as defined in Table 2below. Where amino acids have similar polarity, this can also bedetermined by reference to the hydropathy scale for amino acid sidechains in Table 3.

TABLE 2 Chemical properties of amino acids Ala aliphatic, hydrophobic,neutral Met hydrophobic, neutral Cys polar, hydrophobic, neutral Asnpolar, hydrophilic, neutral Asp polar, hydrophilic, charged Prohydrophobic, neutral (−) Glu polar, hydrophilic, charged Gln polar,hydrophilic, neutral (−) Phe aromatic, hydrophobic, Arg polar,hydrophilic, charged neutral (+) Gly aliphatic, neutral Ser polar,hydrophilic, neutral His aromatic, polar, hydrophilic, Thr polar,hydrophilic, neutral charged (+) Ile aliphatic, hydrophobic, neutral Valaliphatic, hydrophobic, neutral Lys polar, hydrophilic, charged(+) Trparomatic, hydrophobic, neutral Leu aliphatic, hydrophobic, neutral Tyraromatic, polar, hydro- phobic

TABLE 3 Hydropathy scale Side Chain Hydropathy Ile 4.5 Val 4.2 Leu 3.8Phe 2.8 Cys 2.5 Met 1.9 Ala 1.8 Gly −0.4 Thr −0.7 Ser −0.8 Trp −0.9 Tyr−1.3 Pro −1.6 His −3.2 Glu −3.5 Gln −3.5 Asp −3.5 Asn −3.5 Lys −3.9 Arg−4.5

One or more amino acid residues of the amino acid sequence of SEQ ID NO:2 may additionally be deleted from the polypeptides described above. Upto 1, 2, 3, 4, 5, 10, 20 or 30 residues may be deleted, or more.

Variants may include fragments of SEQ ID NO: 2. Such fragments retainpore forming activity. Fragments may be at least 50, 100, 150 or 200amino acids in length. Such fragments may be used to produce the pores.A fragment preferably comprises the pore forming domain of SEQ ID NO: 2.Fragments must include one of residues 88, 90, 91, 105, 118 and 134 ofSEQ ID NO: 2. Typically, fragments include all of residues 88, 90, 91,105, 118 and 134 of SEQ ID NO: 2.

One or more amino acids may be alternatively or additionally added tothe polypeptides described above. An extension may be provided at theamino terminal or carboxy terminal of the amino acid sequence of SEQ IDNO: 2 or polypeptide variant or fragment thereof. The extension may bequite short, for example from 1 to 10 amino acids in length.Alternatively, the extension may be longer, for example up to 50 or 100amino acids. A carrier protein may be fused to an amino acid sequenceaccording to the invention. Other fusion proteins are discussed in moredetail below.

As discussed above, a variant is a polypeptide that has an amino acidsequence which varies from that of SEQ ID NO: 2 and which retains itsability to form a pore. A variant typically contains the regions of SEQID NO: 2 that are responsible for pore formation. The pore formingability of Msp, which contains a β-barrel, is provided by β-sheets ineach subunit. A variant of SEQ ID NO: 2 typically comprises the regionsin SEQ ID) NO: 2 that form β-sheets. One or more modifications can bemade to the regions of SEQ ID NO: 2 that form β-sheets as long as theresulting variant retains its ability to form a pore. A variant of SEQID NO: 2 preferably includes one or more modifications, such assubstitutions, additions or deletions, within its α-helices and/or loopregions.

The monomers derived from Msp may be modified to assist theiridentification or purification, for example by the addition of histidineresidues (a hist tag), aspartic acid residues (an asp tag), astreptavidin tag or a flag tag, or by the addition of a signal sequenceto promote their secretion from a cell where the polypeptide does notnaturally contain such a sequence. An alternative to introducing agenetic tag is to chemically react a tag onto a native or engineeredposition on the pore. An example of this would be to react a gel-shiftreagent to a cysteine engineered on the outside of the pore. This hasbeen demonstrated as a method for separating hemolysin hetero-oligomers(Chem Biol. 1997 July; 4(7):497-505).

The monomer derived from Msp may be labelled with a revealing label. Therevealing label may be any suitable label which allows the pore to bedetected. Suitable labels include, but are not limited to, fluorescentmolecules, radioisotopes, e.g. ¹²⁵I, ³⁵S, enzymes, antibodies, antigens,polynucleotides and ligands such as biotin.

The monomer derived from Msp may also be produced using D-amino acids.For instance, the monomer derived from Msp may comprise a mixture ofL-amino acids and D-amino acids. This is conventional in the art forproducing such proteins or peptides.

The monomer derived from Msp contains one or more specific modificationsto facilitate nucleotide discrimination. The monomer derived from Mspmay also contain other non-specific modifications as long as they do notinterfere with pore formation. A number of non-specific side chainmodifications are known in the art and may be made to the side chains ofthe monomer derived from Msp. Such modifications include, for example,reductive alkylation of amino acids by reaction with an aldehydefollowed by reduction with NaBH₄, amnidination with methylacetimidate oracylation with acetic anhydride.

The monomer derived from Msp can be produced using standard methodsknown in the art. The monomer derived from Msp may be made syntheticallyor by recombinant means. For example, the pore may be synthesized by invitro translation and transcription (IVTT). Suitable methods forproducing pores are discussed in International Application Nos.PCT/GB09/001690 (published as WO 2010/004273), PCT/GB09/001679(published as WO 2010/004265) or PCT/GB10/000133 (published as WO2010/086603). Methods for inserting pores into membranes are discussed.

The transmembrane protein pore is also preferably derived fromα-hemolysin (α-HL). The wild type α-HL pore is formed of seven identicalmonomers or subunits (i.e. it is heptameric). The sequence of onemonomer or subunit of α-hemolysin-NN is shown in SEQ ID NO: 4. Thetransmembrane protein pore preferably comprises seven monomers eachcomprising the sequence shown in SEQ ID NO: 4 or a variant thereof.Amino acids 1, 7 to 21, 31 to 34, 45 to 51, 63 to 66, 72, 92 to 97, 104to 111, 124 to 136, 149 to 153, 160 to 164, 173 to 206, 210 to 213, 217,218, 223 to 228, 236 to 242, 262 to 265, 272 to 274, 287 to 290 and 294of SEQ ID NO: 4 form loop regions. Residues 113 and 147 of SEQ ID NO: 4form part of a constriction of the barrel or channel of α-HL.

In such embodiments, a pore comprising seven proteins or monomers eachcomprising the sequence shown in SEQ ID NO: 4 or a variant thereof arepreferably used in the method of the invention. The seven proteins maybe the same (homoheptamer) or different (heteroheptamer).

A variant of SEQ ID NO: 4 is a protein that has an amino acid sequencewhich varies from that of SEQ ID NO: 4 and which retains its poreforming ability. The ability of a variant to form a pore can be assayedusing any method known in the art. For instance, the variant may beinserted into a lipid bilayer along with other appropriate subunits andits ability to oligomerise to form a pore may be determined. Methods areknown in the art for inserting subunits into membranes, such as lipidbilayers. Suitable methods are discussed above.

The variant may include modifications that facilitate covalentattachment to or interaction with a nucleic acid bind ng protein. Thevariant preferably comprises one or more reactive cysteine residues thatfacilitate attachment to the nucleic acid binding protein. For instance,the variant may include a cysteine at one or more of positions 8, 9, 17,18, 19, 44, 45, 50, 51, 237, 239 and 287 and/or on the amino or carboxyterminus of SEQ ID NO: 4. Preferred variants comprise a substitution ofthe residue at position 8, 9, 17, 237, 239 and 287 of SEQ ID NO: 4 withcysteine (A8C, T9C, N17C, K237C, S239C or E287C). The variant ispreferably any one of the variants described in InternationalApplication No. PCT/GB09/001690 (published as WO 2010/004273),PCT/GB09/001679 (published as WO 2010/004265) or PCT/GB10/000133(published as WO 2010/086603).

The variant may also include modifications that facilitate anyinteraction with nucleotides.

The variant may be a naturally occurring variant which is expressednaturally by an organism, for instance by a Staphylococcus bacterium.Alternatively, the variant may be expressed in vitro or recombinantly bya bacterium such as Escherichia coli. Variants also includenon-naturally occurring variants produced by recombinant technology.Over the entire length of the amino acid sequence of SEQ ID NO: 4, avariant will preferably be at least 50% homologous to that sequencebased on amino acid identity. More preferably, the variant polypeptidemay be at least 55%, at least 60%, at least 65%, at least 70%, at least75%, at least 80%, at least 85%, at least 90% and more preferably atleast 95%, 97% or 99% homologous based on amino acid identity to theamino acid sequence of SEQ ID NO: 4 over the entire sequence. There maybe at least 80%, for example at least 85%, 90% or 95%, amino acididentity over a stretch of 200 or more, for example 230, 250, 270 or 280or more, contiguous amino acids (“hard homology”). Homology can bedetermined as discussed above.

Amino acid substitutions may be made to the amino acid sequence of SEQID NO: 4 in addition to those discussed above, for example up to 1, 2,3, 4, 5, 10, 20 or 30 substitutions. Conservative substitutions may bemade as discussed above.

One or more amino acid residues of the amino acid sequence of SEQ ID NO:4 may additionally be deleted from the polypeptides described above. Upto 1, 2, 3, 4, 5, 10, 20 or 30 residues may be deleted, or more.

Variants may include fragments of SEQ ID NO: 4. Such fragments retainpore-forming activity. Fragments may be at least 50, 100, 200 or 250amino acids in length. A fragment preferably comprises the pore-formingdomain of SEQ ID NO: 4. Fragments typically include residues 119, 121,135. 113 and 139 of SEQ ID NO: 4.

One or more amino acids may be alternatively or additionally added tothe polypeptides described above. An extension may be provided at theamino terminus or carboxy terminus of the amino acid sequence of SEQ ID)NO: 4 or a variant or fragment thereof. The extension may be quiteshort, for example from 1 to 10 amino acids in length. Alternatively,the extension may be longer, for example up to 50 or 100 amino acids. Acarrier protein may be fused to a pore or variant.

As discussed above, a variant of SEQ ID NO: 4 is a subunit that has anamino acid sequence which varies from that of SEQ ID NO: 4 and whichretains its ability to form a pore. A variant typically contains theregions of SEQ ID NO: 4 that are responsible for pore formation. Thepore forming ability of α-HL, which contains a β-barrel, is provided byβ-strands in each subunit. A variant of SEQ ID NO: 4 typically comprisesthe regions in SEQ ID NO: 4 that form β-strands. The amino acids of SEQID NO: 4 that form β-strands are discussed above. One or moremodifications can be made to the regions of SEQ ID NO: 4 that formβ-strands as long as the resulting variant retains its ability to form apore. Specific modifications that can be made to the β-strand regions ofSEQ ID NO: 4 are discussed above.

A variant of SEQ ID NO: 4 preferably includes one or more modifications,such as substitutions, additions or deletions, within its α-helicesand/or loop regions. Amino acids that form α-helices and loops arediscussed above.

The variant may be modified to assist its identification or purificationas discussed above.

Pores derived from α-HL can be made as discussed above with reference topores derived from Msp.

In some embodiments, the transmembrane protein pore is chemicallymodified. The pore can be chemically modified in any way and at anysite. The transmembrane protein pore is preferably chemically modifiedby attachment of a molecule to one or more cysteines (cysteine linkage),attachment of a molecule to one or more lysines, attachment of amolecule to one or more non-natural amino acids, enzyme modification ofan epitope or modification of a terminus. Suitable methods for carryingout such modifications are well-known in the art. The transmembraneprotein pore may be chemically modified by the attachment of anymolecule. For instance, the pore may be chemically modified byattachment of a dye or a fluorophore.

Any number of the monomers in the pore may be chemically modified. Oneor more, such as 2, 3, 4, 5, 6, 7, 8, 9 or 10, of the monomers ispreferably chemically modified as discussed above.

The reactivity of cysteine residues may be enhanced by modification ofthe adjacent residues. For instance, the basic groups of flankingarginine, histidine or lysine residues will change the pKa of thecysteines thiol group to that of the more reactive S⁻ group. Thereactivity of cysteine residues may be protected by thiol protectivegroups such as dTNB. These may be reacted with one or more cysteineresidues of the pore before a linker is attached.

The molecule (with which the pore is chemically modified) may beattached directly to the pore or attached via a linker as disclosed ininternational Application Nos. PCT/GB09/001690 (published as WO2010/004273), PCT/GB09/001679 (published as WO 2010/004265) orPCT/GB10/000133 (published as WO 2010/086603).

Moving

In the method of the invention, the single stranded polynucleotide ismoved through the transmembrane pore. Moving the single strandedpolynucleotide through the transmembrane pore refers to moving thepolynucleotide from one side of the pore to the other. Movement of thesingle stranded polynucleotide through the pore can be driven orcontrolled by potential or enzymatic action or both. The movement can beunidirectional or can allow both backwards and forwards movement.

A polynucleotide binding protein is preferably used to control themovement of the single stranded polynucleotide through the pore. Thisprotein is preferably the same protein that separates the two strands ofthe polynucleotide. More preferably, this protein is Phi29 DNApolymerase. The three modes of Phi29 DNA polymerase, as discussed above,can be used to control the movement of the single strandedpolynucleotide through the pore. Preferably, Phi29 DNA polymeraseseparates the target polynucleotide and controls the movement of theresulting single stranded polynucleotide through the transmembrane pore.

In some embodiments, the entire target polynucleotide (as a singlestranded polynucleotide comprising the one strand of the targetpolynucleotide linked to the other strand of the target polynucleotideby the bridging moiety) will move through the pore. Thus, the entiretarget polynucleotide is moved through the pore and sequenced. In otherembodiments, only part of the target polynucleotide moves through thepore. Such embodiments where only part of the target polynucleotidemoves through the pore may be as follows:

-   -   (i) part of one strand of the target polynucleotide (for example        part of the sense strand of DNA)    -   (ii) all of one strand of the target polynucleotide (for example        all of the sense strand of DNA)    -   (iii) all of one strand (for example all of the sense strand of        DNA) and part of the second strand (for example part of the        anti-sense strand of DNA)

In embodiments where only part of one strand, or all of one strand andpart of the other strand, moves through the pore it is irrelevant whichof the original two strands (i.e. the sense and anti-sense strands) isfully or partially moved through the pore. Furthermore, the order ofmovement of the sense and antisense strands through the pore does notmatter.

In some embodiments, as discussed above and shown in FIG. 13, afterlinking of the two strands of a double stranded analyte and separatingthe two linked strands into a single stranded target polynucleotide, acomplementary strand to the single stranded target polynucleotide isgenerated to form a second construct. The two strands of the secondconstruct may be linked together as described herein. The complementarystrand of the second construct is then separated from the singlestranded target polynucleotide. In this situation, the original singlestranded target polynucleotide may move through the pore and/or thecomplementary strand may move through the pore. In some instances, onlythe complementary strand is sequenced. As described above, this processof separation and complementary strand generation can be repeated asmany times as necessary. This may be referred to herein as the “DUO”method.

When the construct further comprises a leader polymer and a tailpolymer, the single stranded target polynucleotide created afterseparating the two strands of the target polynucleotide preferably movesthrough the pore in the order of: (1) the leader polymer; (2) the onestrand of the target polynucleotide; (3) the bridging moiety; (4) theother strand of the target polynucleotide; and (5) the tail polymer.This is an example of a sequencing a construct made according to the‘MONO’ method.

In an alternative embodiment, a construct produced according to the DUOmethod may pass through the pore in the order of: (1) the leaderpolymer; (2) the first strand of the target polynucleotide; (3) thefirst bridging moiety; (4) the second strand of the targetpolynucleotide; (5) the second bridging moiety; (6) the complement ofthe second strand of the target polynucleotide; (7) the complement ofthe first bridging moiety; (8) the complement of the first strand of thetarget polynucletode and (9) the tail polymer.0

Methods of Sequencing a Double Stranded Target Polynucleotide

The method of the invention comprises moving the single strandedpolynucleotide through a transmembrane pore such that a proportion ofthe nucleotides in the single stranded polynucleotide interact with thepore.

The method may be carried out using any suitable membrane as discussedabove, preferably a lipid bilayer system in which a pore is insertedinto a lipid bilayer. The method is typically carried out using (i) anartificial bilayer comprising a pore, (ii) an isolated,naturally-occurring lipid bilayer comprising a pore, or (iii) a cellhaving a pore inserted therein. The method is preferably carried outusing an artificial lipid bilayer. The bilayer may comprise othertransmembrane and/or intramembrane proteins as well as other moleculesin addition to the pore. Suitable apparatus and conditions are discussedbelow with reference to the sequencing embodiments of the invention. Themethod of the invention is typically carried out in vitro.

The present invention provides methods of sequencing a double strandedtarget polynucleotide. As discussed above, a polynucleotide is amacromolecule comprising two or more pairs of nucleotides. Thenucleotides may be any of those discussed above. The polynucleotide ispreferably a nucleic acid.

These methods are possible because transmembrane protein pores can beused to differentiate nucleotides of similar structure on the basis ofthe different effects they have on the current passing through the pore.Individual nucleotides can be identified at the single molecule levelfrom their current amplitude when they interact with the pore. Thenucleotide is present in the pore if the current flows through the porein a manner specific for the nucleotide (i.e. if a distinctive currentassociated with the nucleotide is detected flowing through the pore).Successive identification of the nucleotides in a target polynucleotideallows the sequence of the polynucleotide to be estimated or determined.

The method comprises (a) providing a construct comprising the targetpolynucleotide, wherein the two strands of the target polynucleotide arelinked by the bridging moiety; (b) separating the two strands of thetarget polynucleotide by contacting the construct with a nucleic acidbinding protein; (c) moving the resulting single stranded polynucleotidethrough the transmembrane pore; and (d) measuring the current passingthrough the pore during each interaction and thereby determining orestimating the sequence of the target polynucleotide. Hence, the methodinvolves transmembrane pore sensing of a proportion of the nucleotidesin a target polynucleotide as the nucleotides individually pass throughthe barrel or channel in order to sequence the target polynucleotide. Asdiscussed above, this is Strand Sequencing.

The whole or only part of the target polynucleotide may be sequencedusing this method. The polynucleotide can be any length. For example,the polynucleotide can be at least 10, at least 50, at least 100, atleast 150, at least 200, at least 250, at least 300, at least 400 or atleast 500 nucleotide pairs in length. The polynucleotide can be 1000 ormore nucleotide pairs, 5000 or more nucleotide pairs or 100000 or morenucleotide pairs in length. The polynucleotide can be naturallyoccurring or artificial. For instance, the method may be used to verifythe sequence of a manufactured oligonucleotide. The methods aretypically carried out in vitro.

The single stranded polynucleotide may interact with the pore on eitherside of the membrane. The single stranded polynucleotide may interactwith the pore in any manner and at any site.

During the interaction between a nucleotide in the single strandedpolynucleotide and the pore, the nucleotide affects the current flowingthrough the pore in a manner specific for that nucleotide. For example,a particular nucleotide will reduce the current flowing through the porefor a particular mean time period and to a particular extent. In otherwords, the current flowing through the pore is distinctive for aparticular nucleotide. Control experiments may be carried out todetermine the effect a particular nucleotide has on the current flowingthrough the pore. Results from carrying out the method of the inventionon a test sample can then be compared with those derived from such acontrol experiment in order to determine or estimate the sequence of thetarget polynucleotide.

The sequencing methods may be carried out using any suitablemembrane/pore system in which a pore is inserted into a membrane. Themethods are typically carried out using a membrane comprisingnaturally-occurring or synthetic lipids. The membrane is typicallyformed in vitro. The methods are preferably not carried out using anisolated, naturally occurring membrane comprising a pore, or a cellexpressing a pore. The methods are preferably carried out using anartificial membrane. The membrane may comprise other transmembraneand/or intramembrane proteins as well as other molecules in addition tothe pore.

The membrane forms a barrier to the flow of ions, nucleotides andpolynucleotides. The membrane is preferably an amphiphilic layer such asa lipid bilayer. Lipid bilayers suitable for use in accordance with theinvention are described above.

The sequencing methods of the invention are typically carried out invitro.

The sequencing methods may be carried out using any apparatus that issuitable for investigating a membrane/pore system in which a pore isinserted into a membrane. The method may be carried out using anyapparatus that is suitable for transmembrane pore sensing. For example,the apparatus comprises a chamber comprising an aqueous solution and abarrier that separates the chamber into two sections. The barrier has anaperture in which the membrane containing the pore is formed.

The sequencing methods may be carried out using the apparatus describedin International Application No. PCT/GB08/000562.

The methods of the invention involve measuring the current passingthrough the pore during interaction with the nucleotide(s). Thereforethe apparatus also comprises an electrical circuit capable of applying apotential and measuring an electrical signal across the membrane andpore. The methods may be carried out using a patch clamp or a voltageclamp. The methods preferably involve the use of a voltage clamp.

The sequencing methods of the invention involve the measuring of acurrent passing through the pore during interaction with the nucleotide.Suitable conditions for measuring ionic currents through transmembraneprotein pores are known in the art and disclosed in the Example. Themethod is typically carried out with a voltage applied across themembrane and pore. The voltage used is typically from −400 mV to +400mV. The voltage used is preferably in a range having a lower limitselected from −400 mV, −300 mV, −200 mV, −150 mV, −100 mV, −50 mV, −20mV and 0 mV and an upper limit independently selected from +10 mV, +20mV, +50 mV, +100 mV, +150 mV, +200 mV, +300 mV and +400 mV. The voltageused is more preferably in the range 100 mV to 240 mV and mostpreferably in the range of 160 mV to 240 mV. It is possible to increasediscrimination between different nucleotides by a pore by using anincreased applied potential.

The sequencing methods are typically carried out in the presence of anyalkali metal chloride salt. In the exemplary apparatus discussed above,the salt is present in the aqueous solution in the chamber. Potassiumchloride (KCl), sodium chloride (NaCl), caesium chloride (CsCl) or amixture of potassium ferrocyanide and potassium ferricyanide istypically used. KCl, NaCl and a mixture of potassium ferrocyanide andpotassium ferricyanide are preferred. The salt concentration may be atsaturation. The salt concentration is typically from 0.1 to 2.5M, from0.3 to 1.9M, from 0.5 to 1.8M, from 0.7 to 1.7M, from 0.9 to 1.6M orfrom 1M to 1.4M. The salt concentration is preferably from 150 mM to 1M.High salt concentrations provide a high signal to noise ratio and allowfor currents indicative of the presence of a nucleotide to be identifiedagainst the background of normal current fluctuations. Lower saltconcentrations may be used if nucleotide detection is carried out in thepresence of an enzyme. This is discussed in more detail below.

The methods are typically carried out in the presence of a buffer. Inthe exemplary apparatus discussed above, the buffer is present in theaqueous solution in the chamber. Any buffer may be used in the method ofthe invention. Typically, the buffer is HEPES. Another suitable bufferis Tris-HCl buffer. The methods are typically carried out at a pH offrom 4.0 to 12.0, from 4.5 to 10.0, from 5.0 to 9.0, from 5.5 to 8.8,from 6.0 to 8.7 or from 7.0 to 8.8 or 7.5 to 8.5. The pH used ispreferably about 7.5.

The methods may be carried out at from 0° C. to 100° C., from 15° C. to95° C., from 16° C. to 90° C., from 17° C. to 85° C., from 18° C. to 80°C., 19° C. to 70° C., or from 20° C. to 60° C. The methods are typicallycarried out at room temperature. The methods are optionally carried outat a temperature that supports enzyme function, such as about 37° C.

As mentioned above, good nucleotide discrimination can be achieved atlow salt concentrations if the temperature is increased. In addition toincreasing the solution temperature, there are a number of otherstrategies that can be employed to increase the conductance of thesolution, while maintaining conditions that are suitable for enzymeactivity. One such strategy is to use the lipid bilayer to divide twodifferent concentrations of salt solution, a low salt concentration ofsalt on the enzyme side and a higher concentration on the opposite side.One example of this approach is to use 200 mM of KCl on the cis side ofthe membrane and 500 mM KCl in the trans chamber. At these conditions,the conductance through the pore is expected to be roughly equivalent to400 mM KCl under normal conditions, and the enzyme only experiences 200mM if placed on the cis side. Another possible benefit of usingasymmetric salt conditions is the osmotic gradient induced across thepore. This net flow of water could be used to pull nucleotides into thepore for detection. A similar effect can be achieved using a neutralosmolyte, such as sucrose, glycerol or PEG. Another possibility is touse a solution with relatively low levels of KCl and rely on anadditional charge carrying species that is less disruptive to enzymeactivity.

The target polynucleotide being analysed can be combined with knownprotecting chemistries to protect the polynucleotide from being actedupon by the binding protein while in the bulk solution. The pore canthen be used to remove the protecting chemistry. This can be achievedeither by using protecting groups that are unhybridised by the pore,binding protein or enzyme under an applied potential (WO 2008/124107) orby using protecting chemistries that are removed by the binding proteinor enzyme when held in close proximity to the pore (J Am Chem Soc. 2010Dec. 22; 132(50):17961-72).

The Strand Sequencing method of the invention uses a polynucleotidebinding protein to separate the two strands of the targetpolynucleotide. More preferably, the polynucleotide binding protein alsocontrols the movement of the target polynucleotide through the pore.Examples of such proteins are given and discussed above.

The two strategies for single strand sequencing are the translocation ofthe single stranded polynucleotide through the transmembrane pore, bothcis to trans and trans to cis, either with or against an appliedpotential. The most advantageous mechanism for strand sequencing is thecontrolled translocation of a single stranded polynucleotide through thenanopore under an applied potential. Exonucleases that act progressivelyor processively on double stranded polynucleotides can be used on thecis side of the pore to feed the remaining single strand through underan applied potential or the trans side under a reverse potential.Likewise, a helicase that unwinds the double stranded polynucleotide canalso be used in a similar manner. There are also possibilities forsequencing applications that require strand translocation against anapplied potential, but the polynucleotide must be first “caught” by theenzyme under a reverse or no potential. With the potential then switchedback following binding the strand will pass cis to trans through thepore and be held in an extended conformation by the current flow. Thesingle strand polynucleotide exonucleases or single strandpolynucleotide dependent polymerases can act as molecular motors to pullthe recently translocated single strand back through the pore in acontrolled stepwise manner, trans to cis, against the applied potential.Alternatively, the single strand DNA dependent polymerases can act asmolecular brake slowing down the movement of a polynucleotide throughthe pore.

In the most preferred embodiment, Strand Sequencing is carried out usinga pore derived from Msp and a Phi29 DNA polymerase. The method comprises(a) providing the double stranded target polynucleotide construct; (b)allowing the target polynucleotide to interact with a Phi29 DNApolymerase, such that the strands are separated and the polymerasecontrols the movement of the target polynucleotide through the Msp poreand a proportion of the nucleotides in the target polynucleotideinteracts with the pore; and (c) measuring the current passing throughthe pore during each interaction and thereby estimating or determiningthe sequence of the target polynucleotide, wherein steps (b) and (c) arecarried out with a voltage applied across the pore.

When the target polynucleotide is contacted with a Phi29 DNA polymeraseand a pore derived from Msp, the target polynucleotide firstly forms acomplex with the Phi29 DNA polymerase. When the voltage is appliedacross the pore, the target polynucleotide/Phi29 DNA polymerase complexforms a complex with the pore and controls the movement of the singlestranded polynucleotide through the pore.

This embodiment has three unexpected advantages. First, the targetpolynucleotide moves through the pore at a rate that is commerciallyviable yet allows effective sequencing. The target polynucleotide movesthrough the Msp pore more quickly than it does through a hermolysinpore. Second, an increased current range is observed as thepolynucleotide moves through the pore allowing the sequence to beestimated or determined more easily. Third, a decreased current varianceis observed when the specific pore and polymerase are used togetherthereby increasing the signal-to-noise ratio.

Any polynucleotide described above may be sequenced.

The pore may be any of the pores discussed above. The pore may compriseeight monomers comprising the sequence shown in SEQ ID NO: 2 or avariant thereof.

As discussed above, wild-type Phi29 DNA polymerase has polymerase andexonuclease activity. It may also unzip double stranded polynucleotidesunder the correct conditions. Hence, the enzyme may work in three modes(as discussed above). The method of the invention preferably involves anMsp pore and Phi29 DNA polymerase. The Phi29 DNA polymerase preferablyseparates the double stranded target polynucleotide and controls themovement of the resulting single stranded polynucleotide through thepore.

Any of the systems, apparatus or conditions discussed above may be usedin accordance with this preferred embodiment. The salt concentration istypically from 0.15M to 0.6M. The salt is preferably KCl.

Kits

The present invention also provides kits for preparing a double strandedtarget polynucleotide for sequencing. The kit comprises (a) a bridgingmoiety capable of linking the two strands of the target polynucleotideand (b) at least one polymer.

In a preferred embodiment, the kit further comprises a leader polymerand a tail polymer. Leader polymers and tail polymers are described indetail above. If the leader and tail polymers are polynucleotides, theleader and tail polymers can be provided as a single unit. In this unit,a portion of the leader polymer and a portion of the tail polymer form adouble strand. This double stranded region may typically be from 5 to 20nucleotide pairs in length. The end of the double stranded portion ofthis unit is linked to the double stranded target polynucleotide.Suitable methods for linking two double stranded polynucleotides areknown in the art. The remainder of the leader and tail polymer remain assingle stranded polynucleotides.

The kit also preferably further comprises one or more markers thatproduce a distinctive current when they interact with a transmembranepore. Such markers are described in detail above.

The kit preferably also comprises means to couple the targetpolynucleotide to a membrane. Means of coupling the targetpolynucleotide to a membrane are described above. The means of couplingpreferably comprises a reactive group. Suitable groups include, but arenot limited to, thiol, cholesterol, lipid and biotin groups.

The kit may further comprise the components of a membrane, such as thephospholipids needed to form a lipid bilayer.

Any of the embodiments discussed above with reference to the methods ofthe invention are equally applicable to the kits of the invention.

The kits of the invention may additionally comprise one or more otherreagents or instruments which enable any of the embodiments mentionedabove to be carried out. Such reagents or instruments include one ormore of the following: suitable buffer(s) (aqueous solutions), means toobtain a sample from a subject (such as a vessel or an instrumentcomprising a needle), means to amplify and/or express polynucleotides, amembrane as defined above or voltage or patch clamp apparatus. Reagentsmay be present in the kit in a dry state such that a fluid sampleresuspends the reagents. The kit may also, optionally, compriseinstructions to enable the kit to be used in the method of the inventionor details regarding which patients the method may be used for. The kitmay, optionally, comprise nucleotides.

Method of Preparing a Target Polynucleotide for Sequencing

The invention also provides a method of preparing a double strandedtarget polynucleotide for sequencing. This method generates theconstruct that allows the target polynucleotide to be sequenced. In thismethod, the two strands of the target polynucleotide are linked by abridging moiety and a polymer is attached to one strand at the other endof the target polynucleotide. The polymer is preferably a leader polymerand the method also preferably further comprises attaching a tailpolymer to the other strand of the target polynucleotide (i.e. at thesame end as the leader polymer). Leader polymers and tail polymers arediscussed in detail above.

The method preferably also further comprises attaching a means to couplethe construct to the membrane to the construct. Such means are describedabove.

The bridging moiety may be synthesized separately and then chemicallyattached or enzymatically ligated to the target polynucleotide. Meansfor doing so are known in the art. Alternatively, the bridging moietymay be generated in the processing of the target polynucleotide. Again,suitable means are known in the art.

A suitable means for preparing a target polynucleotide for sequencing isillustrated in Example 3.

Apparatus

The invention also provides an apparatus for sequencing a doublestranded target polynucleotide. The apparatus comprises (a) a membrane,(b) a plurality of transmembrane pores in the membrane, (c) a pluralityof polynucleotide binding proteins capable of separating the two strandsof the target polynucleotide and (d) instructions for carrying out themethod of the invention. The apparatus may be any conventional apparatusfor polynucleotide analysis, such as an array or a chip. Any of theembodiments discussed above with reference to the methods of theinvention are equally applicable to the kits of the invention.

The apparatus is preferably set up to carry out the method of theinvention.

Suitable nucleic acid binding proteins, such as Phi29 DNA polymerase,are described above.

The apparatus preferably comprises:

a sensor device that is capable of supporting the membrane and pluralityof pores and being operable to perform polynucleotide sequencing usingthe pores and proteins;

at least one reservoir for holding material for performing thesequencing;

a fluidics system configured to controllably supply material from the atleast one reservoir to the sensor device; and

a plurality of containers for receiving respective samples, the fluidicssystem being configured to supply the samples selectively from thecontainers to the sensor device. The apparatus may be any of thosedescribed in International Application No. PCT/GB08/004127 (published asWO 2009/077734), PCT/GB10/000789 (published as WO 2010/122293),International Application No. PCT/GB10/002206 (not yet published) orInternational Application No. PCT/US99/25679 (published as WO 00/28312).

The following Examples illustrate the invention:

EXAMPLE 1 Reading around dsDNA Hairpins

The ability of an enzyme such as Phi29 DNA polymerase to act as amolecular brake along ssDNA, but still also functionally pass alongdsDNA sections, can be exploited to read around the hairpin turn ofdsDNA constructs, permitting DNA/RNA sequencing of both the sense andanti-sense strands. FIG. 3 illustrates how both the sense and anti-sensestrands of dsDNA constructs with hairpin turns can be sequenced with anenzyme such as Phi29 DNA polymerase. In this implementation based onPhi29 DNA polymerase, the dsDNA constructs contain a 5′-ssDNA leader toenable capture under an applied field by a nanopore. This is followed bya dsDNA section that is linked by a hairpin turn. The hairpin turn canoptionally contain a marker (X in FIG. 3) in the turn that creates acharacteristic current signature to aid in identification of the sensestrand region from the anti-sense region. Since the last ˜20bases in thecurrent implementation are not sequenced because the read-head is ˜0.20bases downstrand of the enzyme when it falls off the end of the DNA, theconstructs could also optionally contain a 3′-ssDNA extension to permitreading to the end of the anti-sense region.

The hairpin turns that link the two dsDNA sections could be made of, butare not limited to, sections of DNA/RNA, modified DNA or RNA, PNA, LNA,PEG, other polymer linkers, or short chemical linkers. The hairpinlinkers could be synthesised separately and chemically attached orenzymatically ligated to dsDNA, or could be generated in processing ofthe genomic DNA.

Methods:

DNA: Four separate DNA constructs were prepared as shown in FIGS. 4-7from synthetic DNA (Table 4).

TABLE 4 Synthetic DNA used in experiments of Example 1 ONT NameDNA sequence UZ075′-CCCCCCCCCCCCCCCACCCCCCCCCCCCCCCCCCCTATTCTGTTTATGTTTCTTGTTTGTTAGCCTTTTGGCTAACAAACAAGAAACATAAACACTAATAG-3 (SEQ ID NO: 16) UZ08 5′-CCCCCCCCCCCCCCCACCCCCCCCCCCCCCCCCCCTATTCTGTTTATGTTTCTTG TTTGTTAGCC-3′(SEQ ID NO: 17) UZ12 5′ -GGCTAACAAACAAGAAACATAAACAGAATAG-3′(SEQ ID NO: 18) UA025′-CCCCCCCCCCCCCCCACCCCCCCCCCCCCCCCCCCTATTCTGTTTATGTTTCTTGTTTGTTAGCCTTTTGGCTAACAAACAAGAAACATAAACAGAATAGCCCCCCCCCCTCAGATCTCACTATC-3′ (SEQ ID NO: 19) MS235′-CCCCCCCCCCCCCCCACCCCCCCCCCCCCCCCCCCTATTCTGTTTATGTTTCTTGTTTGTTAGCCTTXTTGGCTAACAAACAAGAAACATAAACACTAATAG-3′ (SEQ ID NO: 20)

All DNA were purchased from Integrated DNA Technologies (IDT) as a PAGEpurified dry pellet, and were resolvated in pure water to a finalconcentration of 100 μM. The short dsDNA construct was prepared byhybridizing UZ08 to UZ12 (Table 4). To hybridize, equal quantities ofthe 100 μM DNA solutions were mixed together, heated to 95° C. on a hotplate, held at 95° C. for 10 min, then allowed to slowly cool to roomtemperature over the course of ˜2 hours. This yields a final solution ofhybridized DNA complex at 50 μM. The UZ07, UA02 and MS23 DNA constructsare hairpins with 4T turns (Table 4). To hybridize the sense andanti-sense regions, the 100 μM DNA solutions were heated to 95° C. on ahot plate, held at 95° C. for 10 min, then rapidly cooled to 4° C. byplacing the samples in a refrigerator. The rapid cooling enhancesintra-molecular hairpin formation over inter-molecular hybridization.The process yields a final solution of hybridized DNA hairpins at 100μM.

MspA production: Purified MspA (NNNRRK) oligomers were made in acell-free Escherichia coli in vitro transcription translation system(Promega). Purified oligomers were obtained by cutting the appropriateoligomer band from a gel after SDS-PAGE, then re-solvating in TE buffer.

Unzipping experiments: Electrical measurements were acquired from singleMspA nanopores inserted in 1,2-diphytanoyl-glycero-3-phosphocholinelipid (Avanti Polar Lipids) bilayers. Bilayers were formed across ˜100μm diameter apertures in 20 μm thick PTFE films (in custom Delrinchambers) via the Montal-Mueller technique, separating two 1 mL bufferedsolutions. All experiments were carried out in a Strand EP buffer of 400mM KCl, 10 mM Hepes, 1 mM EDTA, 1 mM DTT at pH 8.0. Single-channelcurrents were measured on Axopatch 200B amplifiers (Molecular Devices)equipped with 1440A digitizers. Ag/AgCl electrodes were connected to thebuffered solutions so that the cis compartment (to which both nanoporeand enzyme/DNA are added) is connected to the ground of the Axopatchheadstage, and the trans compartment is connected to the activeelectrode of the headstage.

DNA construct and Phi29 DNA polymerase (Enzymnatics, 150 μM) were addedto 100 μL of strand EP buffer and pre-incubated for 5 mins (DNA=1 μM,Enzyme=2 μM). This pre-incubation mix was added to 900 μL of buffer inthe cis compartment of the electrophysiology chamber to initiate captureand unzipping of the complexes in the MspA nanopore (to give finalconcentrations of DNA=0.1 μM, Enzyme=0.2 μM). Only one type of DNA wasadded into the system in a single experiment. Unzipping experiments werecarried out at a constant potential of +180 mV.

Results:

Characteristic and consistent polymerase controlled DNA movements wereobserved when the dsDNA constructs with and without hairpins wereunzipped through MspA nanopores using Phi29 DNA polymerase (FIGS. 4-7).FIGS. 4-7 show the consensus DNA sequence profiles obtained frommultiple single translocations of an analyte through the nanopore) foreach of the DNA constructs shown.

The dsDNA construct (UZ08+UZ12) with no hairpin shows a small number ofsequence dependent states (typically ˜10, FIG. 4). This is consistentwith ˜10-15 bases of the 31 in the dsDNA section passing through theread-head of the nanopore before the enzyme falls off the 3′-end of theDNA (˜20 bases upstrand of the read-head), and the last ˜20 basestranslocate un-braked through the nanopore, too fast to be resolved.

UZ07 (Table 4) contains the same DNA sequence as UZ08+UZ12, but is ahairpin construct with a 4T turn connecting the sense and anti-sensestrands. The consensus sequence obtained from UZ07 (FIG. 5) shares thesame initial profile as UZ08+UZ12, but shows many more sequence states(typically >30) than that for UZ08+UZ12 dsDNA (FIG. 4). This shows thatthe enzyme is proceeding around the hairpin of the sense strand, andalong the anti-sense strand. This allows downstrand reading of theentire sense strand, and part of the anti-sense strand (except the last˜20 bases before the enzyme falls of the 3′-end).

UA02 (Table 4) has the same sequence as UZ07, but with the addition ofan extra 25 bases of non-complementary ssDNA on the 3′-end of theconstruct. The consensus sequence obtained from UA02 (FIG. 6) shows aclosely matching sequence profile to UZ07 (FIG. 5), but with anadditional ˜20 states at the end. The additional 25 bases on the 3′-endpermits the full length of anti-sense to be read before the up-strandenzyme falls off the end of the DNA (˜20 bases upstrand of theread-head). FIG. 8 shows the consensus sequence from UA02—thehomopolymeric 5′-overhang initially in the nanopore (section 1), thesense (section 2), turn (section 3) and antisense (section 4) regions.

Markers can be placed in or near the hairpin turn, that when sequencedcan produce a characteristic signal that permits simple identificationof the sense and anti-sense regions of unknown DNA sequences. MS23(Table 4) has the same sequence as UZ07, but with the addition of anabasic marker in the 4′ turn of the hairpin separating the sense andanti-sense strands. The consensus sequence obtained from MS23 (FIG. 7)shows a closely matching sequence profile to UZ07 (FIG. 5), but with analtered large upwards spike in current in the turn region (marked with*) as a result of the abasic passing through the nanopore read-head atthis point. This large upwards spike in current is characteristic of thereduced ionic blocking of abasic residues in the nanopore constrictionrelative to normal bases, and provides a clear signal by which toseparate the sense and anti sense regions.

Summary:

These experiments demonstrate that Phi29 DNA polymerase is able to readaround the hairpin turn of dsDNA constructs, due to its ability to actas an efficient molecular brake along the ssDNA section.

The read-head of MspA nanopores in this implementation is ˜20basesdownstrand from the DNA at the entrance to the Phi29 enzyme. As aresult, when the enzyme gets to the end of a DNA strand and releases thesubstrate, the remaining ˜20 bases translocate in an uncontrolled mannerthrough the nanopore too quickly to be resolved/sequenced. However,optional 3′-extensions (in this 5′ to 3′ reading direction) can be addedto the DNA constructs to extend the reading distance, which permits fullsequencing of the entire anti-sense strand.

Markers that produce characteristics current signatures can optionallybe placed in or near the hairpin turn of a DNA construct to aid inidentification of the sense and anti-sense regions of the sequence. Themarkers could be, but are not limited to, unique known sequence motifsof normal bases, or unnatural or modified bases that produce alternativecurrent signatures.

EXAMPLE 2 Reading around dsDNA Hairpins on Genomic DNA

Reading around hairpins using Phi29 DNA polymerase can be extended tolong genomic dsDNA with ligated hairpins (FIG. 9). FIG. 9 shows ageneral design outline for creating dsDNA suitable for reading aroundhairpins. The constructs have a leader sequence with optional marker(e.g. abasic DNA) for capture in the nanopore, and hairpin with optionalmarker, and a tail for extended reading into anti-sense strand withoptional marker.

Methods:

DNA: A 400 base-pair section of PhiX 174 RF1 genomic DNA was amplifiedusing PCR primers containing defined restriction sites. Following KasIand MluI restrict ion endonuclease digestion of the 400 bp fragment, DNAadapters containing complimentary ends were then ligated (FIG. 10). Thedesired product was finally isolated by PAGE purification and quantifiedby absorbance at A260 nm.

For ease of analysis each adapter piece contained set abasic markers sothat the progress of the DNA through the nanopore could be tracked. Thesequences of all primers and adapters are given in Table 5

TABLE 5Primer and adapter DNA for creating the genomic hairpin DNA constructs inExample 2. Abasic DNA bases (abasic =X) in the adapters provide markers foreasily identifying the start of the sense strand, the hairpin turn, and theend of the anti-sense strand. ONT Name DNA sequence KasI Sense5′-TTTTTTTTTTGGCGCCCTGCCGTTTCTGATAAGTTGCTT-3′ (SEQ ID NO: 21) PrimerMluI Antisense 5′-AAAAAAAAAAACGCGTAAACCTGCTGTTGCTTGGAAAG-3′(SEQ ID NO: 22) Primer KasI Sense 5′- AdapterTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTXXXXTTTTTTTTTTGGCTAACAAACAAGAAACATAAACAGAATAG-3′ (SEQ ID NO: 23) KasI Antisense5′- Adapter GCGCCTATTCTGTTTATGTTTCTTGTTTGTTAGCCTTTTTTXXXXTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT-3′ (SEQ ID NO: 24) MluI HP 5′-CGCGCTATTCTGTTTATGTTTCTTGTTTGTTAGCCXXXXGGCTAACAAACAAGAAACATAAACAGAATAG (SEQ ID NO: 25)

MspA production: See Example 1.

Unzipping experiments: See Example 1.

The genomic DNA constructs were incubated with Phi29 DNA polymerase(Enzymatics, 150 μM) in 100 μL of strand EP buffer and pre-incubated for5 mins (DNA==5 nM, Enzyme=2 μM). This pre-incubation mix was added to900 μL of buffer in the cis compartment of the electrophysiology chamberto initiate capture and unzipping of the complexes in the MspA nanopore(to give final concentrations of DNA=0.5 nM, Enzyme=0.2 μM). Only onetype of DNA was added into the system in a single experiment. Unzippingexperiments were carried out at a constant potential of +180 mV.

Results:

400mer-No3 (which has a 3′ cholesterol TEG) (Table 6) added to MspAnanopores with Phi29 DNA polymerase resulted in unzipping of the DNA andpolymerase controlled DNA movement lasting 1-3 mins with a large numberof sequence dependent states (FIGS. 11 and 12). The abasic markers, atthe start of the sense strand, in the middle of the hairpin turn, and atthe end of the anti-sense strand, permit easy identification of theseparate sections of the sequence. FIGS. 11 and 12 clearly show 3 abasicpeaks, demonstrating the ability to read around hairpins ligated to longgenomic DNA, and thus sequence both the sense and anti-sense strands ofthe dsDNA.

TABLE 6 Full DNA sequence of genomic construct with ligated adaptersused in the example ONT Name DNA sequence 400 mer No3 5′-TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTXXXXTTTTTTTTTTGGCTAACAAACAAGAAACATAAACAGAATAGGCGCCCTGCCGTTTCTGATAAGTTGCTTGATTTGGTTGGACTTGGTGGCAAGTCTGCCGCTGATAAAGGAAAGGATACTCGTGATTATCTTGCTGCTGCATTTCCTGAGCTTAATGCTTGGGAGCGTGCTGGTGCTGATGCTTCCTCTGCTGGTATGGTTGACGCCGGATTTGAGAATCAAAAAGAGCTTACTAAAATGCAACTGGACAATCAGAAAGAGATTGCCGAGATGCAAAATGAGACTCAAAAAGAGATTGCTGGCATTCAGTCGGCGACTTCACGCCAGAATACGAAAGACCAGGTATATGCACAAAATGAGATGCTTGCTTATCAACAGAAGGAGTCTACTGCTCGCGTTGCGTCTATTATGGAAAACACCAATCTTTCCAAGCAACAGCAGGTTTACGCGCTATTCTGTTTATGTTTCTTGTTTGTTAGCCXXXXGGCTAACAAACAAGAAACATAAACAGAATAGCGCGTAAACCTGCTGTTGCTTGGAAAGATTGGTGTTTTCCATAATAGACGCAACGCGAGCAGTAGACTCCTTCTGTTGATAAGCAAGCATCTCATTTTGTGCATATACCTGGTCTTTCGTATTCTGGCGTGAAGTCGCCGACTGAATGCCAGCAATCTCTTTTTGAGTCTCATTTTGCATCTCGGCAATCTCTTTCTGATTGTCCAGTTGCATTTTAGTAAGCTCTTTTTGATTCTCAAATCCGGCGTCAACCATACCAGCAGAGGAAGCATCAGCACCAGCACGCTCCCAAGCATTAAGCTCAGGAAATGCAGCAGCAAGATAATCACGAGTATCCTTTTCCTTTATCAGCGGCAGACTTGCCACCAAGTCCAACCAAATCAAGCAACTTATCAGAAACGGCAGGGCGCCTATTCTGTTTATGTTTCTTGTTTGTTAGCCTTTTTTXXXXTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT-3′ (SEEQ ID NO: 26)

EXAMPLE 3 Sample Preparation for Sequencing

A 400 base-pair section of PhiX 174 RF1 genomic DNA was amplified usingPCR primers containing defined restriction sites. Following KasI andMluI restriction endonuclease digestion of the 400 bp fragment, DNAadapters containing complimentary ends were then ligated. The desiredproduct was finally isolated by PAGE purification and quantified byabsorbance at A260 nm.

For ease of analysis each adapter piece contained set abasic markers sothat the progress of the DNA through the nanopore could be tracked. Thesequences of all primers and adapters are given below (X=abasicmodification):

KasI Sense Primer (SEQ ID NO: 21)5′-TTTTTTTTTTGGCGCCCTGCCGTTTCTGATAAGTTGCTT-3′ MluI Antisense(SEQ ID NO: 22) 5′-AAAAAAAAAAACGCGTAAACCTGCTGTTGCTTGGAAAG-3′KasI Sense Adapter (SEQ ID NO: 23)5′-TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTXXXXTTTTTTTTTTGGCTAACAAACAAGAAACATAAACAGAATAG- 3′ MluI HP(SEQ ID NO: 25) 5′-CGCGCTATTCTGTTTATGTTTCTTGTTTGTTAGCCXXXXGGCTAACAAACAAGAAACATAAACAGAATAGThe construct is shown in FIG. 10.

If the above adapters contain non-complimentary bases instead of abasicsites then it is possible to open out the analyte using a primercomplimentary to the ssDNA section of the antisense Y-shaped adapter anda strand displacing polymerase (such as Phi29 or Klenow). If the primerand Y-shaped adapter complimentary region also contain a restrictionsite then another hairpin can be ligated, after the amplification, andthe process repeated to expand the template further. This gives theability to sequence the molecule twice; the original sense-antisense ina fully dsDNA unzipping mode and then the amplified sense-antisensestrand in fully ssDNA unzipping mode.

Ligation of DNA adapters to target DNA has been well describedpreviously and this is still the most widespread technique. Ligation ofthe adapters could occur either by single strand ligation using T4 RNAligase I or by double strand ligation of annealed adapters using T4 DNAligase. To prevent target dimer and adapter dimer formation the targetcan be first dA-tailed using a polymerase such as Klenow exo-.

More recently, advances with artificial transposons (such as the Nexterasystem) have begun to show promise in rapidly speeding up adapterattachment while also simultaneously fragmenting the DNA. In theory asimilar approach might be achieved using homologous recombination, suchas the Cre LoxP system (NEB), providing compatible sequences lie withinthe DNA. Advances in chemical ligation have also improved recently,demonstrated with highest success by the successful amplification of DNAstrands containing an unnatural triazole linkage (Sagheer and Brown,2009). For chemical ligation the modification of either the 5′ or 3′ ofthe DNA is usually first required to include a suitable reactive group.Groups can be easily added to the 3′ using a modified dNTP and terminaltransferase. Modification of the 5′ end of DNA has also beendemonstrated but this has so far been limited to thiol groups usingusing T4 Polynucleotide kinase. Some success for direct coupling ofmolecules to the 5′ of DNA via chemical means has been demonstratedusing carbodiimide coupling and such kits are commercially available.However side products are a frequent problem with this chemistry.

EXAMPLE 4 Reading around dsDNA Hairpins of Genomic DNA Using a Helicase

Reading around DNA strands, which consist of connected long genomicdsDNA ligated by hairpins, using a helicase enzyme was investigated(FIG. 17). The constructs used have a leader sequence with optionalmarker (e.g. abasic DNA) for capture in the nanopore, a hairpin withoptional marker, and a tail which has an extended reading sequence and acholesterol tether attached to the end.

Methods:

To link the sense and antisense strands a bridging hairpin (SEQ ID NO:32) was ligated to one end. A synthetic Y-adaptor was ligated on toallow enzyme binding and threading into the nanopore: the sense strand(SEQ ID NO: 29 attached to SEQ ID NO: 30 via four abasic DNA bases, seeFIG. 18) of this adaptor contains the 5′ leader, a sequence that iscomplementary to the tether sequence (SEQ ID NO: 35, which at the 3′ endof the sequence has six iSpl8 spacers attached to two thymine residuesand a 3′ cholesterol TEG) and 4 abasics. The antisense half of theadaptor also has a 3′ hairpin which will act as an intramolecular primerfor later conversion to a. DUO analyte (SEQ ID NO: 31, see FIG. 22starter template). MONO analyte DNA was prepared using a ˜400 bp regionof PhiX 174 (Sense strand sequence=SEQ ID NO: 33 and antisense strandsequence=SEQ ID NO: 34). The region of interest was PCR amplified withprimers containing SacI and KpnI restriction sites (SEQ ID NO's: 27 and28 respectively). Purified PCR product was then SacI and KpnI digestedbefore aY-shaped adapter (sense strand sequence (SEQ ID NO: 29 attachedto SEQ ID NO: 30 via four abasic DNA bases) is ligated onto the 5′ endof SEQ ID NO: 33 and the anti-sense strand (SEQ ID NO: 31) is ligatedonto the 3′ end of the SEQ ID NO: 34) and a hairpin (SEQ ID NO: 32, usedto join SEQ ID NO's: 33 and 34) were ligated to either end, using T4 DNAligase (See FIG. 18 for final DNA construct). The product was purifiedfrom a 5% TBE PAGE gel and eluted by crush and soak method into TEbuffer.

MspA production: Purified MspA oligomers of the mutant MspA poreMS(B1-G75S-G77S-L88N-Q126R)8 MspA (SEQ ID NO: 2 with the mutationsG75S/G77S/L88N/Q126R) were made in a cell-free Escherichia coli in vitrotranscription translation system (Promega). Purified oligomers wereobtained by cutting the appropriate oligomer band from a gel afterSDS-PAGE, then re-solvating in TE buffer.

Helicase experiments—Electrical measurements were acquired from singleMspA nanopores (MS(B1-G75S-G77S-L88N-Q126R)8 MspA (SEQ ID NO: 2 with themutations G75S/G77S/L88N/Q126R)) inserted in1,2-diphytanoyl-glycero-3-phosphocholine lipid (Avanti Polar Lipids)bilayers. Bilayers were formed across ˜100 μm diameter apertures in 20μm thick PTFE films (in custom Delrin chambers) via the Montal-Muellertechnique, separating two 1 mL buffered solutions. All experiments werecarried out in a buffer of 400 mM NaCl, 100 mM Hepes, 10 mM potassiumferrocyanide, 10 mM potassium ferricyanide, pH8.0, at an appliedpotential of +140 mV. Single-channel currents were measured on Axopatch200B amplifiers (Molecular Devices) equipped with 1440A digitizers.Platinum electrodes were connected to the buffered solutions so that thecis compartment (to which both nanopore and enzyme/DNA are added) isconnected to the ground of the Axopatch headstage, and the transcompartment is connected to the active electrode of the headstage.

A single pore was obtained before MgCl2 and dTTP were added to the cischamber to give final concentrations of 10 mM and 5 mM respectively.Data was obtained for 5 mins at +140 mV before DNA (SEQ ID NOs: 29-35connected as shown in FIG. 18) was added to the cis chamber for a finalconcentration of 0.1 nM and data obtained for a further 5 mins. Helicasewas added to the cis chamber to a final concentration of 100 nM and anyhelicase controlled DNA movements were recorded at +140 mV.

Results:

The 400 bp sense/antisense hairpin construct (SEQ ID NO's: 29-35connected as shown in FIG. 18) when added to an MspA nanopore(MS(B1-G75S-G77S-L88N-Q126R)8 MspA (SEQ ID NO: 2 with the mutationsG75S/077S/L88N/Q126R)) with a helicase resulted in unzipping of the DNAand helicase controlled DNA movement, with a large number of sequencedependent states (FIGS. 19 to 21). The 400 bp sense/antisense hairpinconstruct (SEQ ID NO's: 29-35 connected as shown in FIG. 18) producedhelicase controlled DNA movement that permitted easy identification ofthe start of the sequence, as the polyT region and the abasic DNA basesat the start of the sense strand can be observed (highlighted with a *and a # respectively in FIG. 20). Therefore, it was possible to showthat the helicase could control the movement and unzipping of a 400 bphairpin. The clear change in speed, between the sense and antisenseregions, highlights the point where the enzyme passed around the cornerand is a useful marker between these regions (FIG. 21, the change fromreading the sense region (1) to reading the anti-sense region (2) isshown with a *). This alteration in speed eliminates the need formarkers to mark the hairpin turn. This demonstrates the ability to readaround hairpins ligated to long genomic DNA with a helicase, and thussequence both the sense and anti-sense strands of the dsDNA.

EXAMPLE 5 Production of DUO Polynucleotide Hairpin Strands

It has been demonstrated already that linking the information from thesense and the antisense strands is possible by ligating a synthetichairpin to one end of the DNA. This serves to give a read of the naturalsense and antisense strands from one molecule at the same time, somaking base-calling more accurate as one gets two chances to call asingle position.

To link the sense and antisense strands a bridging hairpin (SEQ ID NO:32) can be ligated to one end. It is also possible to ligate on asynthetic Y-adaptor to allow enzyme binding and threading into thenanopore: the sense strand (SEQ ID NO: 29 attached to SEQ ID NO: 30 viafour abasic DNA bases, see FIG. 22 starter template) of this adaptorcontains the 5′ leader, a sequence that is complementary to the tethersequence (SEQ ID NO: 35, which at the 3′ end of the sequence has sixiSp18 spacers attached to two thymine residues and a 3′ cholesterol TEG)and 4 abasics, the antisense half of the adaptor also has a 3′ hairpin(SEQ ID NO: 31) which will act as an intramolecular primer for laterconversion to a DUO analyte (see FIG. 22 starter template).

When the bridging hairpin (SEQ ID NO: 32) is ligated it is also possibleto ligate on a synthetic Y-adaptor: the sense strand (SEQ ID NO: 29attached to SEQ ID NO: 30 via four abasic DNA bases, see FIG. 22 startertemplate) of this adaptor contains the 5′ leader, a sequence that iscomplementary to the tether sequence (SEQ ID NO: 35, which at the 3′ endof the sequence has six iSp18 spacers attached to two thymine residuesand a 3′ cholesterol TEG) and 4 abasics, and the antisense half of theadaptor has a 3′ hairpin which will act as an intramolecular primer (SEQID NO: 31, see FIG. 22 starter template) this affords us the opportunityto further expand the template by copying the entire, now linked senseand antisense, using a strand displacing polymerase that binds to the 3′end of the Y-adaptor (Step 2 of FIG. 22). The Y-shaped and hairpinadaptors contain mis-matched restriction sites (not sensitive torestriction digest, see top of FIG. 23). When the analyte issubsequently filled-in and expanded (see FIG. 22 steps 2 to 3), therestriction sites are completed (See bottom of FIG. 23), therefore, thefully filled-in analyte (SEQ ID NO: 29-36 connected as shown in FIG. 25)can be digested using site specific restriction endonucleases to confirmsuccessful fill-in.

DUO analyte was prepared from the MONO analyte disclosed in Example 4above. The doubly ligated MONO PAGE purified analyte (SEQ ID NO's: 29-35connected as shown in FIG. 18) was further incubated with Klenow DNApolymerase, SSB and nucleotides to allow extension from the Y-shapedadapter hairpin (SEQ ID NO: 31). To screen for successful DUO product(SEQ D NOs: 29-36 connected as shown in FIG. 25) a series of mismatchrestriction sites were incorporated into the adapter sequences, wherebythe enzyme will cut the analyte only if the restriction site has beensuccessfully replicated by the DUO extension process (See FIG. 23, MONOanalyte at the top and DUO analyte at the bottom).

FIG. 24 shows that the adapter modified analyte (MONO, SEQ ID NO: 29-35)in the absence of polymerase does not digest with the restrictionenzymes (see gel on the left in FIG. 24, Key: M=MfeI, A=AgeI, X=XmaI,N=NgoMIV, B=BspEI), due to the fact they are mismatched to one another,as shown in FIG. 23 top. However, on incubation with polymerase there isa noticeable size shift and the shifted product (DUO) now digests asexpected with each of the restriction enzymes (see gel on the right inFIG. 24, Key: M=MfeI, A=AgeI, X=XmaI, N=NgoMIV, B=BspEI). This showsthat using the described method it is possible to produce DUO product(SEQ ID NOs: 29-36 connected as shown in FIG. 25).

EXAMPLE 6 Reading around dsDNA DUO Polynucleotide Hairpins Using aHelicase

Reading around DUO hairpins constructs (SEQ ID NO's: 29-36 connected asshown in FIG. 25), which consist of original sense (SEQ ID NO: 33) andanti-sense strands (SEQ ID NO: 34) as well as replicate sense andreplicate strands (SEQ ID NO: 36), using a helicase enzyme wasinvestigated.

Methods: The DNA construct used in this experiment was produced by themethod disclosed in Example 5 above.

MspA production: The MspA pore MS(B1-G75S-G77S-L88N-Q126R)8 MspA (SEQ IDNO: 2 with the mutations G75S/G77S/L88N/Q126R) was produced by themethod described in Example 4.

Unzipping experiments Electrical measurements were acquired, asdescribed in Example 4, from single MspA nanopores(MS(B1-G75S-G77S-L88N-Q126R)8 MspA (SEQ ID NO: 2 with the mutationsG75S/G77S/L88N/Q126R)) inserted in1,2-diphytanoyl-glycero-3-phosphocholine lipid (Avanti Polar Lipids)bilayers in buffer solution (400 mM NaCl, 100 mM Hepes, 10 mM potassiumferrocyanide, 10 mM potassium ferricyanide, pH8.0) at an appliedpotential of +140 mV.

Initially, MgCl2 (10 mM) and dTTP (5 mM) were added to the ciscompartment and a control experiment run for 5 mins. Secondly, the DNAconstruct (0.1 nN, SEQ ID NOs: 29-36 connected as shown in FIG. 25) wasadded to the cis compartment and a further control experiment run for 5mins. Finally, the helicase (100 nM) was added to the electrophysiologychamber to initiate helicase activity. All unzipping experiments werecarried out at a constant potential of +140 mV.

Results:

The DUO hairpin construct (SEQ ID NOs: 29-36 connected as shown in FIG.25) when added to an MspA nanopore (MS(B1-G75S-G77S-L88N-Q126R)8 MspA(SEQ ID NO: 2 with the mutations G75S/G77S/L88N/Q126R)) with a helicaseresulted in unzipping of the DNA and helicase controlled DNA movement,with a large number of sequence dependent states (FIGS. 26 to 28). FIG.26 shows two typical helicase controlled DNA movements, the regionswhich correspond to the original sense section, original antisensesection, the replicate sense region and the replicate antisense sectionsare labeled 1 to 4 respectively. FIG. 27 shows a magnified view of oneof the helicase controlled DNA movement from FIG. 26 and FIG. 28 showsanother magnified view of the transition between the original sense andanti sense strands. The change in speed between the helicase controllingthe movement of the sense strand in comparison to the antisense strandis clearly visible (FIGS. 26-28). This alteration in speed eliminatesthe need for markers to mark the hairpin turn. This demonstrates theability to read around DUO hairpin constructs (SEQ ID NOs: 29-36connected as shown in FIG. 25), and thus sequence both the sense andanti-sense strands of the dsDNA twice. This makes base-calling moreaccurate as one gets four chances to call a single position.

The invention claimed is:
 1. A method for nanopore sensing, the methodcomprising: i) coupling a nucleic acid construct transiently to amembrane via a 3′ cholesterol TEG tether, wherein the membrane comprisesan amphiphilic layer that is a 1,2-diphytanoyl-glycero-3-phosphocholinelipid bilayer, and wherein a MspA transmembrane pore is present in themembrane, wherein the MspA transmembrane pore is a nanopore, and isconfigured to permit flow of ions through the transmembrane pore fromone side of the membrane to the other side of the membrane, wherein theconstruct comprises a target polynucleotide having a target portion, aleader, and the 3′ cholesterol TEG tether, wherein the construct isconfigured such that the leader facilitates engagement of the constructwith the transmembrane pore while the construct is transiently coupledto the membrane, promoting passage of the nucleic acid construct throughthe nanopore, and wherein the step of transiently coupling the constructto the membrane comprises contacting the membrane with the construct,wherein the construct comprises, prior to the contacting step, thetarget polynucleotide, the leader, and the 3′ cholesterol TEG tether;and ii) sensing at least the target portion of the target polynucleotideupon movement of the construct through the nanopore, wherein sensingcomprises reading nucleotides of the nucleic acid construct as thenucleotides pass through the nanopore in order to sequence the targetportion, wherein the nucleotides are read from single channel currentsobtained from electrodes connected to a buffered solution on cis andtrans sides of the lipid bilayer comprising the nanopore.